The organoarsenical roxarsone is added to poultry feed to increase weight gain. Studies have shown that roxarsone does not accumulate in poultry tissue but is excreted, resulting in elevated arsenic concentrations (~40 mg/kg) in poultry litter. However, there is little understanding of the fate of roxarsone once it is introduced into agricultural watersheds. Using batch experiments, I investigated the sorption characteristics of roxarsone to Ap and Bt soils of the Frederick series, common in the Shenandoah Valley of Virginia, an area of intense poultry production. Results demonstrate that roxarsone sorbs strongly to Bt soils, but only showed moderate to low sorption onto Ap soils. Sorption to the Ap soils demonstrated stronger pH dependence than did sorption to the Bt soils. Removing organic matter (OM) from Ap soils significantly changed the sorption characteristics, suggesting that OM may be coating mineral surfaces in these soils. Results of this study have implications for roxarsone transport in agricultural watersheds. For soils that have had years of poultry litter application, there will be both sorption and subsequent leaching of roxarsone. In the OM-rich Ap horizon, OM controls sorption. Because roxarsone is loosely bound to OM, it would be rapidly leached into water after a recharge event or field irrigation. Once roxarsone reaches the Bt soils, it is strongly sorbed into iron oxides or clays, decreasing the potential for leaching. However, competition from phosphate or organic acids for sorption sites on mineral surfaces may affect roxarsone retention in the Bt soils. / Master of Science

This study investigated the adsorption properties of roxarsone, an organoarsenic poultry feed additive, to goethite and kaolinite in order to determine what role mineral surfaces play in controlling the mobility of roxarsone in watersheds where poultry litter is applied. Adsorption edge experiments for goethite and kaolinite showed a dependence on pH for both As(V) and roxarsone. This pattern can be explained by the pH-dependent changes in the mineral surface charge and protonation of the aqueous arsenic species. Isotherms for As(V) and roxarsone on goethite and kaolinite show surface saturation for As(V), but not for roxarsone. The overall adsorption patterns show that As(V) and roxarsone adsorption is similar, suggesting that the arsenate functional group is the dominant control on roxarsone adsorption. However, there are some subtle differences between adsorption of As(V) and roxarsone, which can be explained by the relative sizes of the molecules, the presence of functional groups, differences in solubility, and differences in the type of adsorption (monolayer versus multilayer). Comparison of roxarsone adsorption to goethite and kaolinite reveals that at the low concentrations of roxarsone that are expected to leach from poultry litter into soil water, goethite adsorbs roxarsone more strongly then kaolinite. However, due to the abundance of kaolinite, both are important controls on roxarsone mobility. / Master of Science

A study was conducted to investigate the effects of roxarsone and Bio-Mos® on broiler production, gut morphology and bone strength. Three thousand and ten broilers were randomly assigned to 1 of 5 dietary corn-soybean meal based treatments: 1) negative control (NEG), basal diet; 2) positive control (POS; NEG + 27 ppm Bacitracin MD); 3) roxarsone (ROX; POS + 50 ppm of roxarsone); 4) Bio-Mos® (BIO; POS + 0.15 and 0.5% Bio-Mos® added during the starter and grower periods, respectively); 5) Bio-Mos®+All-Lac XCL (BIO+LAC; POS + 0.2, 0.1, and 0.05% Bio-Mos® during the starter, grower and finishing periods, respectively and 0.25g All-Lac XCL/bird sprayed at hatchery). On day 14, 7 of the 14 replicate pens/treatment were challenged with Eimeria maxima (3 x 104 oocysts/bird). Tibias were collected on day 28 and 49 to determine bone-breaking strength. Non-challenged birds had higher body weight gains (BWG) and lower feed conversion (FCR) from day 0 to 49 than challenged birds (P < 0.05). Jejunal crypt depth was increased in challenged broilers compared to non-challenged broilers at 28 days-of-age (P < 0.05). From day 0 to 35, ROX birds had lower BWG and FI than BIO and BIO+LAC birds (P < 0.05), while FCR was similar. Supplementing roxarsone resulted in reduced feed intake and BWG, but no significant differences were noted in FCR compared to feeding Bio-Mos®. ROX fed broilers had decreased ileal crypt depth compared to all other dietary treatments (P < 0.05). Muscle As concentration was lower than FDA allowable limits in broilers fed ROX without a withdrawal period at 28 days-of-age. Including roxarsone or Bio-Mos® did not generally improve production compared to broilers fed the negative diet. / Master of Science

Several types of antibiotics (roxarsone, virginiamycin, and bacitracin) are widely included in poultry feed to improve animal growth yields. Most of the antibiotics are excreted in manure which is subsequently applied to soils. One concern with this practice is that antibiotics may affect several microbially-mediated nutrient cycling reactions in soils that influence crop productivity and water quality. The main objectives of this study were to determine the effects of livestock antibiotics on nitrification, denitrification, and microbial community composition in soils along a topographic gradient. These objectives were addressed in a series of lab experiments by monitoring changes in inorganic N species and ester-linked fatty acid methyl ester profiles after exposing soil microorganisms collected from different topographic positions to increasing levels of antibiotics. It was discovered that roxarsone and virginiamycin inhibited nitrification and soil microbial growth and also influenced microbial community composition, but only at levels that were much higher than expected in poultry litter-applied soils. Bacitracin did not affect nitrification, microbial growth, or microbial community composition at any concentration tested. None of the antibiotics had a strong affect on denitrification. Thus, it is unlikely that soil, water, or air quality would be significantly impacted by the antibiotics contained in poultry litter.

Bovine serum albumin (BSA) is the most abundant protein in plasma and it is responsible for the transport of most of the exogenous and endogenous compounds present in blood. Roxarsone (RX) and its metabolite acetarsone (AC) are organic compounds of arsenic(V) used in poultry as a food additive. In this sense, the interaction process of RX and AC compounds with BSA was evaluated under physiological conditions using spectroscopic techniques. It was possible to determine the formation of a supramolecular complex between RX and AC ligands using molecular fluorescence. The interaction degree was moderate, with a binding constant (Kb) of 4.27x105 L mol-1 and 0,27x105 L mol-1 for RX and AC, respectively. The quenching mechanism involved in the interaction process for both ligands was static and, preferably, by electrostatic interactions to the BSA-RX complex and hydrogen bonds and Van der Waals forces to BSA-AC. Both the formation of the complex and the quenching mechanism were confirmed by UV-vis spectroscopy. The 3D fluorescence analysis evidenced structural changes in the BSA polypeptide chain, being more pronounced in the presence of the RX ligand in comparison to AC. In addition, the BSA-RX interaction process modified the BSA surface, leading to reduction of hydrophobic regions more effectively compared to BSA-AC. About the binding site, both ligands are preferably in the site II of BSA. From the results obtained by 1H NMR, greater chemical shift was observed in the presence of the AC ligand, being the region close to amide group interacting with BSA, while the RX ligand possibly interacts more superficially in the protein. Thus, the RX and AC ligands do not favor the kinetics of protein fibrillation, giving indications that the possible deleterious effects in the evaluated in vitro system are not related to this process. Alkaline phosphatase inhibition kinetics demonstrated inhibition of enzymatic activity from 5 min and signal stabilization in 55 min of analysis for both RX and arsenate (positive control). Finally, the evaluation of in vitro alkaline phosphatase activity showed inhibition of enzymatic activity in 42% in the presence of the RX ligand and 5% in the AC ligand, giving indications of more detrimental effects for RX when compared to AC. / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / A albumina do soro bovino (BSA) é a proteína mais abundante do plasma e responsável pelo transporte da maioria dos compostos exógenos e endógenos presentes no sangue. Roxarsone (RX) e seu metabólito acetarsone (AC) são compostos orgânicos de arsênio(V) empregados na avicultura como aditivo alimentar. Neste sentido, o processo de interação dos compostos RX e AC com a BSA foi avaliado sob condições fisiológicas utilizando técnicas espectroscópicas. Empregando fluorescência molecular foi possível determinar a formação do complexo supramolecular para os ligantes RX e AC. O grau de interação foi moderado, com constante de ligação (Kb) de 4,27x105 L mol-1 e 0,27x105 L mol-1 para RX e AC, respectivamente. O tipo de quenching envolvido no processo de interação para ambos os ligantes foi estático e, preferencialmente, por interações de natureza eletrostática para o complexo BSA-RX e ligações de hidrogênio e forças de Van der Waals para BSA-AC.Tanto a formação do complexo quanto o tipo de quenching foram confirmados por espectroscopia de UV-vis. A análise por fluorescência 3D evidenciou mudanças estruturais na cadeia polipeptídica da BSA, sendo mais pronunciadas na presença do ligante RX em comparação ao AC. Ademais, o processo de interação BSA-RX modificou a superfície da BSA, levando à redução de regiões hidrofóbicas de forma mais efetiva em comparação à BSA-AC. Com relação ao sítio de ligação, ambos os ligantes encontram-se preferencialmente no sítio II da BSA. A partir dos resultados obtidos por RMN 1H observou-se maior deslocamento químico na presença do ligante AC, estando a porção próxima ao grupamento amida interagindo com a BSA. Enquanto que o ligante RX possivelmente interage mais superficialmente na proteína. Além disso, os ligantes RX e AC não favorecem a cinética de fibrilação protéica, dando indícios que os possíveis efeitos deletérios no sistema in vitro avaliado não estão relacionados a este processo. A cinética de inibição da fostatase alcalina demonstrou inibição da atividade enzimática a partir de 5 min e estabilização do sinal em 55 min de análise tanto para o RX quanto para o arseniato (controle positivo). Por fim, a avaliação da inibição da atividade da fosfatase alcalina in vitro evidenciou inibição da atividade enzimática em 42% na presença do ligante RX e 5% para o ligante AC, dando indícios de efeitos mais prejudiciais para o RX quando comparado ao AC.

Organoarsenicals such as methylarsenical methylarsenate (MAs(V)) and aromatic arsenicals including roxarsone (4-hydroxy-3-nitrophenylarsenate or Rox(V)) have been extensively used as an herbicide and growth enhancers in animal husbandry, respectively. They undergo environmental degradation to more toxic inorganic arsenite (As(III)) that contaminates crops and drinking water. We previously identified a bacterial gene (arsI) responsible for aerobic MAs(III) demethylation. The gene product, ArsI, is a Fe(II)-dependent extradiol dioxygenase that cleaves the carbon-arsenic (C-As) bond in MAs(III) and trivalent aromatic arsenicals. The objective of this study was to elucidate the ArsI mechanism. Using isothermal titration calorimetry, we determined the dissociation constants (Kd) and ligand-to-protein stoichiometries (N) of ArsI for Fe(II), MAs(III) and aromatic phenyl arsenite. Using a combination of methods including chemical modification, site-directed mutagenesis, and fluorescent spectroscopy, we demonstrated that amino acid residues predicted to participate in Fe(II)-binding (His5-His62-Glu115) and substrate binding (Cys96-Cys97) are all involved in catalysis. Finally, the products of Rox(III) degradation were identified as As(III) and 4-hydroxy-2-nitrophenol, demonstrating that ArsI is a dioxygenase that incorporates one oxygen atom from dioxygen into the carbon and the other to the arsenic to catalyze the cleavage of the C-As bond. These results augment our understanding of the mechanism of this novel C-As lyase.