Arsenic Metabolites, Including N

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Arsenic metabolites, including N-acetyl-4-hydroxy-m-arsanilic acid, in chicken litter from a Roxarsone-feeding study involving 1600 chickens Zonglin Yang, Hanyong Peng, Xiufen Lu, Qingqing Liu, Bin Hu, Rongfu Huang, Gary Kachanoski, Martin J. Zuidhof, and X. Chris Le Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.5b05619 • Publication Date (Web): 13 Feb 2016 Downloaded from http://pubs.acs.org on February 13, 2016

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Environmental Science & Technology is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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Environmental Science & Technology

Roxarsone

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Arsenic metabolites, including N-acetyl-4-hydroxy-m-arsanilic acid, in chicken litter from a Roxarsone-feeding study involving 1600 chickens

Zonglin Yang,† Hanyong Peng,‡ φ Xiufen Lu,‡ Qingqing Liu,‡ Rongfu Huang,‡ Bin Hu,φ Gary Kachanoski,# Martin Zuidhof,§ and X. Chris Le *, †, ‡ †

Department of Chemistry and ‡Department of Laboratory Medicine and Pathology, University

of Alberta, 10-102 Clinical Sciences Building, Edmonton, Alberta, Canada T6G 2G3 φ

Department of Chemistry, Wuhan University, Wuhan, China

#

Department of Renewable Resources, University of Alberta, Edmonton, Alberta, Canada

§

Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton,

Alberta, Canada T6G 2P5

*

Corresponding author. [email protected] (X. Chris Le)

1 ACS Paragon Plus Environment

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Abstract The poultry industry has used organoarsenicals, such as 3-nitro-4-hydroxyphenylarsonic acid (Roxarsone®, ROX), to prevent disease and to promote growth. Although previous studies have analyzed arsenic species in chicken litter after composting or after application to agricultural lands, it is not clear what arsenic species were excreted by chickens before biotransformation of arsenic species during composting. We describe here the identification and quantitation of arsenic species in chicken litter repeatedly collected on days 14, 24, 28, 30, and 35 of a Roxarsone-feeding study involving 1600 chickens of two strains. High performance liquid chromatography separation with simultaneous detection by both inductively coupled plasma mass spectrometry and electrospray ionization tandem mass spectrometry provided complementary information necessary for the identification and quantitation of arsenic species. A new metabolite, N-acetyl-4-hydroxy-m-arsanilic acid (N-AHAA), was identified, and it accounted for 3-12% of total arsenic. Speciation analyses of litter samples collected from ROXfed chickens on days 14, 24, 28, 30, and 35 showed the presence of N-AHAA, 3-amino-4hydroxyphenylarsonic acid (3-AHPAA), inorganic arsenite (AsIII), arsenate (AsV), monomethylarsonic acid (MMAV), dimethylarsinic acid (DMAV), and ROX. 3-AHPAA accounted for 3-19% of the total arsenic. Inorganic arsenicals (the sum of AsIII and AsV) comprised 2-6% (mean 3.5%) of total arsenic. Our results on the detection of inorganic arsenicals, methylarsenicals, 3-AHPAA, and N-AHAA in the chicken litter support recent findings that ROX is actually metabolized by the chicken or its gut microbiome. The presence of the toxic metabolites in chicken litter is environmentally relevant as chicken litter is commonly used as fertilizers.



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Introduction 3-nitro-4-hydroxyphenylarsonic acid (Roxarsone®, ROX) has been well known as the first approved arsenic-containing drug commonly used in poultry industry, until June 2011 when the manufacturer of ROX, Pfizer subsidiary Alpharma, announced discontinuing the sale of ROX in poultry feed in the United States[1]. Nitarsone, a similar organoarsenical drug, continues to be sold and used by the turkey industry; and ROX is still in use in Asia. ROX is typically added to poultry feed to promote weight gain, improve feed efficiency, and control intestinal parasites that cause coccidiosis [2]. Although the ROX ingested by chickens was previously thought to be mainly excreted unchanged in waste, Moody and Williams[3] have observed metabolism of ROX to 3-amino-4-hydroxyphenylarsonic acid (3-AHPAA). Furthermore, higher levels of inorganic arsenic (arsenate and arsenite) were recently detected in the livers[4,5] and muscle tissue [6,7 ] of chickens fed ROX than in untreated chickens. Each year an estimated 12 to 23 billion kilograms (kg) of poultry litter is produced in the U.S. [8]. Garbarino et al. [9] estimated that the annual chicken litter could contain 250,000 kg of arsenic, if 70% of the 8.6 billion chickens raised in the U.S. were fed a diet containing 45.4 g of ROX per ton of the diet [10]. Up to 90% of the poultry litter in the U.S. is subsequently disposed of to agricultural lands as a fertilizer [11], and arsenic may be taken up by crops grown in arsenicamended fields [12-14]. Poultry litter has been fed to beef cattle as an economical source of proteins, minerals, and energy [15,16]; prior to the year 2000, 20-25% of the waste was used for feed in Virginia [16]. Several companies in the U.K. and the U.S. also utilize poultry litter as electrical and heating fuels [17]. Because the toxicity of arsenic varies greatly with arsenic species [18]

, it is critical to identify and quantify the concentration of individual arsenic species.


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Several studies [9, 19, 20, 21] have shown that ROX is the dominant arsenic species in poultry litter, with minor amounts of arsenate (AsV), arsenite (AsIII), monomethylarsonic acid (MMAV), and dimethylarsinic acid (DMAV) also detectable. There is also evidence that ROX in chicken litter is partly biotransformed by microorganisms to inorganic AsV [9, 19] and 3-amino-4hydroxyphenylarsonic acid (3-AHPAA) [19, 21]. However, it is not clear whether the transformation products of ROX were excreted by chickens or formed during the composting of the chicken manure, because most of these studies analyzed chicken manure during or after composting. There is no study reporting the determination of arsenic species excreted by chickens over a controlled feeding period. The excretion of individual arsenic species from chickens may vary over feeding time; but there is no quantitative information regarding temporal changes of these arsenicals. We report here the speciation of arsenic in the litter of chickens fed a ROX-supplemented diet over a 35-day period. The inclusion of 1600 chickens makes this the largest feeding study designed to understand the excretion, metabolism, distribution, and update of arsenic. Analysis of arsenic species in samples collected on days 14, 24, 28, 30, and 35 enabled observation of temporal changes of individual arsenic species.

Experimental Section Reagents. 3-nitro-4-hydroxyphenylarsonic acid (98.1% purity, Sigma-Aldrich, St. Louis, MO), sodium m-arsenite (97.0%, Sigma, St. Louis, MO), sodium arsenate (99.4%, Sigma), monosodium acid methane arsonate (99.0%, Chem Service, West Chester, PA), cacodylic acid (98%, Sigma), and arsenobetaine (98%, Tri Chemical Laboratories Inc., Japan) were used to prepare respectively ROX, AsIII, AsV, MMAV, DMAV and AsB stock solutions containing 1000



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mg As/L in 18.2 MΩ·cm deionized water [22]. 3-amino-4-hydroxyphenylarsonic acid (3-AHPAA) and N-acetyl-4-hydroxy-m-arsanilic acid (N-AHAA, or acetarsone) were purchased from Pfaltz and Bauer, Inc. (Waterbury, CT). Both stock solutions at 100 mg As/L were prepared by dissolving their purified solids in deionized water with 5% HPLC-grade methanol (Fisher Scientific, Fair Lawn, NJ). Concentrations of arsenic in these arsenic stock solutions were determined using ICPMS, against a primary calibration standard of inorganic arsenic solution (Agilent Technologies, Santa Clara, CA). Standard reference material (SRM) 1640a trace elements in natural water was obtained from the National Institute of Standards and Technology (Gaithersburg, MD), and was used as a quality control measure of total arsenic determination. Stock solutions were kept at 4 °C prior to the time of use. Arsenic standard solutions for speciation analysis were prepared daily from stock solutions using deionized water as the diluent. Feeding Experiments. A Roxarsone-feeding study was conducted at the Poultry Research Centre, University of Alberta. A total of 1600 chickens, representing two commercial broiler strains, Ross 308 and Cobb 500, were used. For each strain, 400 chickens were placed in 4 pens (100 chickens in each pen) to serve as the control group, and another 400 chickens were raised in 4 additional pens (also 100 chickens per pen) to serve as the ROX-treatment group. Drinking water from the same source (

Arsenic Metabolites, Including N

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