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Public Health Risk of Arsenic Species in Chicken Tissues from Live Poultry Markets of Guangdong Province, China Yuanan Hu, Wenfeng Zhang, Hefa Cheng, and Shu Tao Environ. Sci. Technol., Just Accepted Manuscript • Publication Date (Web): 21 Feb 2017 Downloaded from http://pubs.acs.org on February 21, 2017
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Public Health Risk of Arsenic Species in Chicken Tissues
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from Live Poultry Markets of Guangdong Province, China
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Yuanan Hu1, Wenfeng Zhang2, Hefa Cheng3*, Shu Tao3
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1 MOE Laboratory of Groundwater Circulation and Evolution
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School of Water Resources and Environment
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China University of Geosciences (Beijing)
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Beijing 100083, China
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2 State Key Laboratory of Organic Geochemistry
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Guangzhou Institute of Geochemistry, Chinese Academy of Sciences
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Guangzhou 510640, China
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3 MOE Key Laboratory for Earth Surface Processes
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College of Urban and Environmental Sciences
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Peking University
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Beijing 100871, China
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Submitted to: Environmental Science & Technology
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* Corresponding author phone: (+86) 10 6276 1070; fax: (+86) 10 6276 7921; e-mail:
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[email protected] ACS Paragon Plus 1 Environment
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Abstract
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Arsenic-based feed additives, such as roxarsone (ROX), are still legally and widely used in food
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animal production in many countries.
This study was conducted to systematically characterize
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the content and speciation of arsenic in chicken tissues from live poultry markets and in
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commercial chicken feeds in Guangdong, a major poultry production and consumption province in
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China, and to assess the corresponding public health risk.
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commercial feeds could be modeled as a mixture of two log-normal distributions (geometric
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means: 0.66 and 17.5 mg/kg), and inorganic arsenic occurred at high levels (0.19-9.7 mg/kg) in
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those with ROX detected. In general, chicken livers had much higher contents of total arsenic
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compared to the muscle tissues (breast and drumstick), and chicken muscle from the urban
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markets contained arsenic at much higher levels than that from the rural markets.
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incremental lifetime cancer risk (bladder and lung cancer) from dietary exposure to arsenic
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contained in chicken meat products on local markets was above the serious or priority level (10-4)
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for 70 and 30% of the adult populations in Guangzhou and Lianzhou, respectively.
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findings indicate the significant need to phase out the use of arsenic-based feed additives in China.
The total arsenic contents in the
The
These
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1. Introduction
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Phenylarsenic compounds, including roxarsone (ROX) and p-arsanilic acid (p-ASA), had been
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widely used as feed additives in poultry and swine production around the world over the past
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several decades, primarily for the purposes of controlling coccidial parasites, promoting weight
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gain, and enhancing meat pigmentation.1,2 Although ROX, which is used primarily in poultry
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production, is of low toxicity, its routine use can lead to residues of more toxic inorganic arsenic
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species in chicken tissues.3-9
Exposure to inorganic arsenic (i-As) can result in increased risk of ACS Paragon Plus 3 Environment
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bladder, lung, and skin cancer, as well as several non-cancer diseases, including cardiovascular
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disease, diabetes, and cognitive deficits.10-13 With residues of arsenic compounds potentially
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occurring at elevated levels in chicken tissues, health risk could arise from consumption of such
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meat products.1,14,15
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1999,16 while the U.S. Food and Drug Administration (FDA) phased out their use in 2013.17
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Nonetheless, they are still widely used in many countries around the world, including Austrlia,
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Brazil, China, and India.18-20
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even after the recent ban in the U.S. due to the fast expansion of factory farming.21,22 For ROX,
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an allowable dose limit of 50 mg/kg in chicken feed has been set in China,23 but little information
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is available on its consumption in poultry production.
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commercial chicken feeds on the markets in Guangdong province were supplemented with ROX,
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with contents ranging from 0.7 to 17.7 mg/kg.22
The European Union banned the use of arsenic-based feed additives in
In particular, the use of ROX has shown a growing trend in China
A recent study found that 30% of
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The residues and speciation of arsenic in animal meat products resulting from the use of
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arsenic-based feed additives in swine and poultry farming have received little attention until
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recently.6,24 A study by the U.S. FDA found that the use of ROX as a feed additive increased the
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levels of various arsenic species, such as As(III), As(V), dimethylarsinic acid (DMA), and
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monomethylarsonic acid (MMA), in the tissues of broilers.9 A market basket study conducted in
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the U.S. before the use of ROX was banned showed that meat products of chickens raised in
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conventional farms had much higher contents of i-As compared to those of antimicrobial-free
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chickens raised in conventional farms and organic chickens.6
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additional bladder and lung cancer cases per 100,000 people could result from lifetime
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consumption of the conventional chicken meat products compared with consumption of organic
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chickens in the U.S.6
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It was further estimated that 3.7
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There is a significant need to understand the health risk of arsenic residues in chicken tissues
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associated with the use of arsenic-based feed additives in chicken production in China.
The
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present study aimed to assess the public health risk of dietary exposure to the arsenic species
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present in chicken tissues on live poultry markets in Guangdong province, which ranks among the
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top in both chicken production and consumption per capita in China. The results show that the
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chicken tissues on both urban and rural markets contained elevated levels of arsenic species, and
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dietary intake of i-As from lifetime consumption of chicken meat products could pose significant
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cancer risk to local populations.
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2. Methods
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2.1. Sample collection and analysis
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A total of 354 samples of chicken tissues were randomly purchased from 9 live poultry markets
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in Guangzhou and 5 live poultry markets in Lianzhou of Guangdong province (Supporting
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Information or SI Figure 1), between January 2013 and October 2014 (see SI Table 1 for details).
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All samples were collected fresh (within 1 h of slaughtering), and were taken to the laboratory (in
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Guangzhou) or a -20 °C refrigerator (in Lianzhou) in coolers within 1 h. The samples from
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Lianzhou were subsequently transported in frozen state to the laboratory in Guangzhou. Once
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received in the laboratory, all samples (defreezed if necessary) were rinsed with distilled water,
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and sliced into small pieces. They were powderized after being freeze-dried and then stored at
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-20 °C prior to sample preparation and analysis (within 2 weeks).
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Consumers in Guangdong prefer live poultry over frozen meat, and consider the former to be much healthier and fresher.
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at different times, covering those consumed by the local populations.
These samples were also
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expected to be generally representative of the products sold on local food markets.
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one of the most developed urban centers in China, while Lianzhou, which has a per capita gross
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domestic product (GDP) only ~1/6 of that of Guangzhou (127,800 Yuan in 2014), is a relatively
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under-developed area in Guangdong province.
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Guangzhou were mostly supplied by factory farms, while those on the Lianzhou markets came
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largely from family and small farms, according to the poultry vendors.
Guangzhou is
Chickens on the live poultry markets in
126 127
A total of 61 samples of commercial chicken feeds covering all major types and brands, which
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were collected from retailer markets across Guangdong province in November 2011, were kindly
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supplied by South China Agricultural University (see SI Table 2 for details).
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large-scale poultry farms rely exclusively on commercial feeds in their production, while chickens
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raised in backyard and small family farms are often fed with corn, bran, and kitchen leftover.
Mid- and
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The total contents of arsenic in the feed and tissue samples were determined by inductively
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coupled plasma-mass spectrometry (ICP-MS) after microwave-assisted digestion, while the major
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inorganic and organic arsenic species, including arsenobetaine (AsB), DMA, MMA, i-As, p-ASA,
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and ROX, in selected tissue and feed samples were extracted using microwave-assisted extraction
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(MAE), and then detected with high performance liquid chromatography hyphenated with ICP-MS
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(HPLC-ICP-MS), using the method developed in our group.25 The method detection limits were
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0.936 g/kg for AsB, 4.68 g/kg for i-As, 3.12 g/kg for DMA, 4.68 g/kg for MMA, 8.06 g/kg
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for p-ASA, and 18.7 g/kg for ROX in chicken tissues (wet weight, as As), respectively.
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of sample preparation, digestion and extraction methods, instrumental analysis, and quality
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assurance and quality control procedures followed are summarized in the SI.
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2.2. Data analysis
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Fisher’s Least Significant Difference (LSD) test was utilized to identify whether the contents of
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arsenic had statistically significant difference between various chicken tissues, and unpaired t-test
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was applied to determine if the samples collected from Guangzhou and Lianzhou had significant
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difference. Pearson’s correlation analysis was performed to evaluate the relationship between the
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total arsenic contents in different tissues of the same chickens and the association among the
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various arsenic species in chicken livers.
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Due to lack of predictive models for the risk of cancer and non-cancer diseases in Chinese
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population, we applied the methods developed by the U.S. Environmental Protection Agency
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(USEPA) to assess the corresponding health risk of arsenic exposure associated with chicken
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consumption.
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The lifetime average daily dose (LADD) of i-As was estimated as: 𝐿𝐴𝐷𝐷 =
𝐶𝑖𝐴𝑠 ×𝐼𝑅
(1)
𝐵𝑊
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where CiAs is the content of i-As in chicken tissue (mg/kg, wet weight), IR is the lifetime per capita
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daily intake rate of chicken tissue (wet weight), and BW is the average body weight. The IR for
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populations of Guangzhou and Lianzhou were estimated from the provincial average consumption
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rate of rural residents, and the expenditures on poultry purchases of urban households in
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Guangdong, respectively (SI Table 3).
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ingestion was calculated as:
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The incremental lifetime cancer risk (ILCR) of arsenic
𝑅𝑐 = 𝐿𝐴𝐷𝐷 × 𝑞 ∗
(2)
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where Rc is the cancer risk, and q* is the cancer slope factor for i-As. The Integrated Risk
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Information System (IRIS) (last revised in 1998) lists a q* value of 1.5 (mg/kg BW/day)-1 for i-As,
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which corresponds to skin cancer.26
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BW/day)-1 for bladder and lung cancer based on epidemiologic literatures.27
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susceptibility is well-known to depend on ethnicity and region, but no specific data on Chinese
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population for arsenic exposure is available. Thus, the q* values established from the U.S.
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population were adjusted with an ethnicity factor of 0.86, which had been used in estimating the
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lung cancer risk of polycyclic aromatic hydrocarbon exposure for Asian populations.28
In 2010, the USEPA proposed a q* value of 25.7 (mg/kg The cancer
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The non-carcinogenic risk of arsenic ingestion was estimated as: 𝐻𝑄 =
𝐴𝐷𝐷
(3)
𝑅𝑓𝐷
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where HQ is the hazard quotient, ADD is the average daily dose and is approximated as LADD
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here, and RfD is the reference dose for oral exposure.
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i-As, which is recommended by the IRIS27, was adopted here.
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below 1.0, while non-carcinogenic effects may occur when it exceeds this threshold.
A RfD value of 3×10-4 mg/kg BW/day for No significant risk exists at HQ
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The human variability in genetic polymorphism and other uncertainty factors in the risk
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estimation were accounted for by Monte Carlo simulation.
The total arsenic contents in chicken
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tissues were observed to follow log-normal distribution, and the means and standard deviations
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(STDs) of the log-transformed data were used in the simulation.
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arsenic content was assumed to follow a normal distribution with a mean of 0.3 and a STD of 0.15,
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as discussed in Section 3.4.
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distributions, with means of 48.8 and 25.2 g/day per person for adults in Guangzhou and Lianzhou,
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respectively (SI Table 3).
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study in Guangdong province.29
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59.5 and 57.2 kg, respectively, and the estimated STDs of log-transformed body weights are 0.15
The fraction of i-As in the total
The chicken consumption rates were also assumed to follow normal
The STDs were assumed to be 70% of the means, according to a recent The mean body weights for Guangzhou and Lianzhou adults are
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and 0.16, respectively, based on the results of a national survey.30 The uncertainty of cancer
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slope factors and reference dose for oral As exposure was accounted for based on the USEPA’s
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documents.26,27
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and lung cancer, and log-transformed RfD were assumed to be 0.16, 0.25, and 0.5, respectively.
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The genetic susceptibility of cancer risk was adjusted with a factor that was assumed to follow
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log-normal distribution with a mean of 1 and log-transformed deviation of 0.65.28 Based on the
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probability distributions of the above parameters, the ILCR and HQ of Guangzhou and Lianzhou
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populations from consumption of chicken tissues on local markets were assessed with Monte
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Carlo simulation (10,000 runs using randomly sampled values each time).31
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independent and identical simulation results could well represent the probability distributions of
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the estimated ICLR and HQ.
The STDs of log-transformed q* for skin cancer, log-transformed q* for bladder
The large number of
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3. Results and Discussion
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3.1. Total contents and speciation of As in commercial chicken feeds
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Figure 1a depicts the empirical cumulative distribution function for the content of total As in the
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61 feed samples. The total As contents in the commercial feeds varied largely (0.30-33.3 mg/kg),
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with a geometric mean (GM) of 1.56 mg/kg.
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As contents exceeding 2.0 mg/kg, which is the national standard for total arsenic in animal feeds,
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although this value actually refers to i-As.32
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samples contained total As above 10.0 mg/kg, and near 5% of them had >30.0 mg/kg of total As.
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Such high levels of total As in the chicken feeds are indicative of the supplementation of
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arsenic-based feed additives.
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the total As content of the feed samples and the fit by a binary mixture model.
Over 30% of the commercial feed samples had total
Furthermore, approximately a quarter of the feed
Figure 1b shows the histogram and probability density function for
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It appears that the
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distribution of total As content in the commercial feeds could be described as a mixture of two
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log-normal distributions with GM values of 0.66 and 17.5 mg/kg, respectively, which probably
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correspond to the feeds without and with ROX supplementation. Around 70% of the commercial
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feeds had total As contents below 1.8 mg/kg, which were probably not supplemented with
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arsenic-based feed additives.
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Due to budgetary constraints, the contents of major inorganic and organic arsenic species
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present in the commercial feeds were analyzed in a total of 18 samples randomly selected from the
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61 samples (Table 1). ROX was not detected in 2/3 of the samples analyzed, which is consistent
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with the relatively low total As contents observed in most commercial feeds. In the 6 feed
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samples with ROX detected, its content ranged from 0.10 to 16.1 mg/kg, with a GM of 1.78 mg/kg.
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The GM of total As contents in these samples was 4.84 mg/kg (range: 0.30 to 18.7 mg/kg), while
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that in the feed samples without apparent ROX detection was only 0.41 mg/kg (range: 0.20 to 0.57
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mg/kg).
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higher contents of i-As (GM: 2.48 mg/kg) compared to those without apparent ROX detection
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(GM: 0.39 mg/kg), indicating significant contamination of the commercial feeds by inorganic
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arsenic compounds. In addition, i-As accounted for more than half of the total As contents in
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most of the feed samples with ROX detected (5 out of 6). The elevated levels of i-As probably
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occurred as an impurity (i.e., unreacted inorganic arsenic compounds) from the production of ROX.
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A nationwide survey of animal feeds conducted in 2012 found that the feed products from 34
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manufacturers did not meet the safety requirements, and around half of them contained arsenic
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exceeding the allowable limit.33 The inorganic arsenic might also result from degradation of
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ROX in the feeds during storage and handling, as a variety of microbes are capable of degrading
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ROX,34-38 although the actual contribution of this deserves further investigation.
It is worth noting that the feed samples with measurable ROX contents also had much
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According to the hygiene standard for animal feeds in China, the maximum allowable dose of
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ROX in chicken feeds is 14.2 mg/kg as As (50 mg/kg as ROX).23
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feed samples are mostly in compliance with this limit. However, significant portion of the
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commercial feeds with ROX detection also contained i-As at levels above the allowable limit (2
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mg/kg), while none of the samples without detectable ROX had >2.0 mg/kg of i-As.
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survey in Guangdong province also reported that elevated levels of i-As occurred in the chicken
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feed samples with ROX detection.22
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particularly i-As, can greatly elevate the potential of arsenic accumulation in the bodies of farmed
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animals, which not only exerts toxic effect on the animals, but also poses direct health risk to the
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consumers of animal meat products.
The contents of ROX in the
A recent
Feed products with unsafe levels of arsenic species,
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3.2. Total contents of As in chicken tissues
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Figure 2 shows the empirical cumulative distribution functions for the total As contents in
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various types of chicken tissues (liver, gizzard, heart, kidney, breast, and drumstick).
Results of
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LSD test indicate that the total contents of As in livers and gizzards were significantly higher than
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those in the muscle tissues (breast and drumstick) (SI Table 4). In general, livers contained the
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highest levels of total As, with 11 of the 124 liver samples (8.9%) having >0.5 mg/kg of total
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arsenic (wet weight), which is the upper limit allowed for meat products in China.39 In contrast,
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none of the 110 muscle samples (breast and drumstick) contained total As above this limit.
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results are consistent with the previous findings that livers had the highest contents of arsenic
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among chicken tissues.5,9,40
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significant positive correlations with those in the other tissues (drumstick, heart, kidney, and
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gizzard) of the same chickens (SI Table 5). It is also noted that the total As contents in chicken
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tissues from Guangdong, China were comparable with those reported in the U.S. before the
These
The contents of total arsenic in livers were also found to have
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phase-out of arsenic-based feed additives in poultry production (SI Table 4).
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Table 2 compares the arsenic contents in chicken tissue samples collected from the live poultry
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markets in Guangzhou and Lianzhou.
In general, the giblet and muscle samples from Guangzhou
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had higher levels of total As than those from Lianzhou.
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the muscle samples from these two locations showed statistically significant difference (27.1 vs.
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15.6 g/kg). This probably resulted from the wide use of arsenic-based feed additives in factory
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farms, which were the dominant source for chickens on the markets in Guangzhou. In contrast,
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traditional backyard farms and small family farms still contributed largely to the chicken supply in
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the predominantly rural and economically under-developed Lianzhou area.
In particular, the GM of As contents in
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3.3. Speciation of As in chicken tissues
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The toxicity of arsenic compounds varies significantly, with inorganic species (i.e., As(III) and
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As(V)) being much more toxic than organoarsenicals.41,42 Thus chemical speciation of arsenic in
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the chicken tissues could provide important information on their health risk.
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analysis was further carried out for 24 tissue samples with relatively high levels of total arsenic.
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Figure 3 shows the HPLC-ICP-MS chromatograms of AsB, MMA, DMA, i-As, ROX, p-ASA in
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the extracts of selected chicken liver samples, in comparison with that of a mixed standard.
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occurrence of ROX in the chicken tissues is indicative of food-borne residues resulting from its
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administration during farming.
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which is consistent with the fact that not all commercial feeds had been supplemented with
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arsenic-based feed additives.
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be oxidized to As(V) during MAE with an alkaline medium (pH 10.0) and subsequent sample
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processing in open air. Thus, the contents of i-As in the tissue samples were quantified from the
Arsenic speciation
The
The peak for ROX was absent on one of the chromatograms,
It is worth noting that As(III) in the tissue samples was expected to
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peaks of As(V), which could be partially contributed by the As(III) originally present in the tissues.
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Multiple unidentified arsenic species were also detected in the chicken liver samples analyzed.
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Although ROX fed to the chickens is mostly excreted in unchanged from, it could be metabolized
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in
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N-acetyl-4-hydroxyphenylarsonic acid (N-AHPAA) and 4-amino-phenylarsonic acid (4-APAA)
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are two possible metabolites of ROX that have been detected in the livers of chickens treated with
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ROX.43,46 N-AHPAA and 4-APAA could well be among the unidentified arsenic species based
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on preliminary comparison of the elution orders of various arsenic species with those reported by
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Peng and co-workers.46 Nonetheless, further refinement of our analytical method and validation
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with authentic standards are necessary to confirm this.
the
liver,43
and
possibly
degraded
by
the
gut
microbiota.34,44,45
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Table 3 summarizes the contents of major inorganic and organic arsenic species detected in the In general, ROX was detected as the major arsenic species (GM: 57.4 g/kg),
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24 tissue samples.
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followed by unidentified As species (GM: 57.0 g/kg) and i-As (GM: 52.4 g/kg), in the liver
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samples, while the contents of ROX were below the detection limit in the muscle and gizzard
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samples.
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detected in most of the samples, including all livers.
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p-ASA was not detected in any of the feed samples analyzed.
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particular sample probably resulted from the misuse of feed additives. The relatively low levels
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of DMA and MMA in the tissues could be attributed to metabolism of the inorganic and organic
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arsenic compounds ingested from drinking water and chicken feeds. Studies have shown that
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chicken and mammalian gut microbiota can convert i-As to DMA and MMA.45,47
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frequently detected in chicken livers (15 out of 18), while DMA occurred in all the muscle and
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gizzard samples.
p-ASA was detected in only one tissue sample, while unidentified arsenic species were Used primarily as an additive in swine feed,
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Thus its occurrence in the
MMA was
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Table 3 also compares the contents of various arsenic species detected in the chicken tissue
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samples from the live poultry markets in Guangdong with those reported in the literature.
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Nachman and co-workers reported that the GM values for the contents of i-As and DMA in the
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raw meats of chickens farmed in different ways (organic vs. conventional, and with unknown or
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prohibiting policy on the use of arsenic-based feed additives) on the U.S. markets (before the
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phase-out of ROX) were 0.7 and 2.7 g/kg, respectively.6
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contents of corresponding arsenic species detected in the chicken tissues from Guangdong
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province. In a 35-day feeding study, Liu and co-workers found that i-As occurred at a mean
324
content of 3.1 g/kg in the breast of chickens treated with ROX,4 which is comparable to the level
325
we observed.
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both control and treatment groups in the study of Liu and co-workers,4 was only detected at low
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levels in some of the tissue sample.
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North America was caused by the prevalent use of fish meal as a protein source in chicken feeds.4
329
In contrast, its low occurrence in the chicken tissues in this study could be explained by the fact
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that the chicken feeds in China were primarily made of corn and soybean meal, with only minor
331
portion of the proteins coming from animals (including fish meal), especially for the finisher feeds.
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AsB is a non-toxic organoarsenical that occurs commonly in marine animals, and in diverse
333
non-marine organisms as well.48-50 Although consumption of AsB from various food sources
334
contributes significantly to the aggregate arsenic exposure, little risk to human health is expected
335
due to its low toxicity in humans (LD50>104 mg/kg body weight).49,50
These values are much lower than the
It is noted that AsB, which is widely observed in the chicken breast samples from
The frequent occurrence of AsB in the chicken meats from
336 337
Pearson’s correlation analysis showed a moderate positive correlation between i-As and ROX
338
(coefficient of 0.40 and p-value of 0.05) in the chicken livers (SI Table 6), indicating that the
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inorganic arsenic species might originate from ROX metabolites and/or other ROX-related sources
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(e.g., elevated levels of i-As found in the feeds with ROX detection).
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coefficient of 0.81 (p-value