Transfer of arsenic from poultry feed to poultry litter: A

Science of the Total Environment 630 (2018) 302–307

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Science of the Total Environment journal homepage: www.elsevier.com/locate/scitotenv

Transfer of arsenic from poultry feed to poultry litter: A mass balance study Sanjay K. Gupta a, X. Chris Le c, Gary Kachanosky a, Martin J. Zuidhof b, Tariq Siddique a,⁎ a b c

Department of Renewable Resources, University of Alberta, Edmonton, AB T6G2G7, Canada Department of Agricultural Food and Nutritional Sciences, University of Alberta, Edmonton, AB T6G2P5, Canada Division of Analytical and Environmental Toxicology, University of Alberta, Edmonton, AB T6G2G3, Canada

H I G H L I G H T S

G R A P H I C A L

A B S T R A C T

• First study that describes mass balance of arsenic intake and excretion by chickens • Two strains Ross and Cobb exhibited similar response to roxarsone-amended feed. • Arsenic did not retain in chickens during their growth on arsenic containing feed. • Poultry litter enriched in arsenic warrants investigations if used in crop production.

a r t i c l e

i n f o

Article history: Received 1 January 2018 Received in revised form 8 February 2018 Accepted 10 February 2018 Available online xxxx Editor: Jay Gan Keywords: Roxarsone Arsenic Poultry feed Arsenic intake Arsenic excretion Poultry litter Arsenic recovery

a b s t r a c t Roxarsone (rox), an arsenic (As) containing organic compound, is a common feed additive used in poultry production. To determine if As present in rox is excreted into the poultry litter without any retention in chicken meat for safe human consumption, the transference of As from the feed to poultry excreta was assessed using two commercial chicken strains fed with and without dietary rox. The results revealed that both the strains had similar behaviour in growth (chicken weight; 2.17–2.25 kg), feed consumption (282–300 kg pen−1 initially containing 102 chicken) and poultry litter production (73–81 kg pen−1) during the growth phase of 35 days. Our mass balance calculations showed that chickens ingested 2669–2730 mg As with the feed and excreted out 2362–2896 mg As in poultry litter during the growth period of 28 days when As containing feed was used, yielding As recovery between 86 and 108%. Though our complementary studies show that residual arsenic species in rox-fed chicken meat may have relevance to human exposure, insignificant retention of total As in the chicken meat substantiates our mass balance results. The results are important in evaluating the fate of feed additive used in poultry production and its potential environmental implications if As containing poultry litter is applied to soil for crop production. © 2018 Elsevier B.V. All rights reserved.

1. Introduction ⁎ Corresponding author. E-mail addresses: [email protected] (X.C. Le), [email protected] (G. Kachanosky), [email protected] (M.J. Zuidhof), [email protected] (T. Siddique).

https://doi.org/10.1016/j.scitotenv.2018.02.123 0048-9697/© 2018 Elsevier B.V. All rights reserved.

Arsenic (As) is a toxic element in the environment and a known carcinogen that consistently ranks first on the Agency for Toxic Substances and Disease Registry (ASTDR) Substance Priority List (https://www.

S.K. Gupta et al. / Science of the Total Environment 630 (2018) 302–307

atsdr.cdc.gov/spl/index.html). Sources of As contamination in the environment includes its elevated contents in some earth crust minerals, industrial activities (mining and smelting) and arsenical compounds used for different purposes (ATSDR, 2007). Drinking water and ingestion of contaminated food are primary As exposure pathways to biological receptors (humans and animals) that pose a significant threat to public health (Naujokas et al., 2013). Roxarsone (rox; 3-nitro-4-hydroxyphenylarsonc acid), an organic compound containing As, was added routinely to the poultry feed for parasitic disease prevention, growth promotion, enhanced feed utilization and improved meat pigmentation for N60 years (Nachman et al., 2013; Fisher et al., 2015) until its use was discontinued in Europe (European Commission, 1999), USA (FDA, 2013), and Canada (https:// www.theglobeandmail.com/life/health-and-fitness/sales-halted-afterarsenic-found-in-chicken-drug/article591962/). When the United States Food and Drug Administration (FDA) approved rox in 1944 as a feed additive (Nachman et al., 2013), it was believed that the nontoxic organic As present in rox would not change into toxic inorganic As inside the chicken body; rather organic As would be excreted unchanged into the poultry manure leaving chicken meat safe for human consumption (Schmidt, 2012). However, higher than tolerable amounts of total As (N2.0 mg kg−1) established by FDA before 1963 (FDA, 1963) were found in the livers of rox-fed chickens compared to the ones fed without rox (FDA, 2011). Subsequent studies focused on the determination of different species of As in different chicken body parts as the toxicity of As is highly dependent on its chemical species (Moe et al., 2016). Higher concentrations of inorganic As were found in the conventional compared to the anti-biotic free conventional chicken meat samples in the US (Nachman et al., 2013). Similarly, inorganic forms of As was also detected in the feather meal products from six US states (Nachman et al., 2012). Yao et al. (2016) revealed that rox in chicken diet was transformed into its metabolites (different As species) in chicken manures which subsequently increased concentrations of these As species in rice plants (grain, straw and hull) when grown in soil amended with the chicken manures. However, no study to date has reported the mass balance of As from poultry feed to poultry litter. In our study, we conducted an experiment growing two strains of chicken on roxsupplemented feed or feed devoid of rox. Different arsenic species were analyzed in chicken liver (Peng et al., 2014; Peng et al., 2017), chicken breast (Liu et al., 2016) and chicken litter (Yang et al., 2016) samples taken from this study. In the current manuscript, we report mass balance that determines the amount of total As taken in by chickens with feed consumption and the amount of total As excreted in poultry litter to assess the possible retention of As inside the chicken body. Arsenic utilization in poultry industry and its subsequent transport to different components of biosphere may have far-reaching consequences related to human health and the environment. Though rox containing poultry feed has been discontinued in Europe and North America, many other countries continue to use phenylarsenicals in the poultry industry (Nachman et al., 2012; Yao et al., 2016). 2. Methodology 2.1. Poultry feed and chicken growth experiment Effect of two types of feed (rox containing feed: rox, and feed without rox: control) was studied on two commercial strains of poultry (Cobb 500 and Ross 308) grown in an environmentally controlled barn as described previously by Liu et al. (2016). Briefly, in the barn, age related temperature and ventilation protocols were followed as per the Strain Management Guides of Cobb (Cobb-Vantress, 2008) and Ross (Aviagen, 2009). The lighting program followed a photoperiod of 23 h for the first three days, and then 20 h' light and 4 h' dark from fourth day to the end of the experiment (35 days). In total, there were

303

16 pens with a treatment combination of two types of feed, two poultry strains and four replications. In each pen (169 cm × 420 cm), 102 chicks (one-day old, mixed sex, previously weighed) were reared. New softwood shavings of known weight were used as the bedding material in each pen that yielded a depth of 7.5 cm above the pen floor. For the rox treatment, 3-NITRO® (Alpharma Canada Corporation) containing 20% rox was added to the feed at 250 g ton−1. Because rox contains 28.48% As, the As content was 14.24 mg kg−1 of the feed prepared for the rox treatment. The feed used for the control treatment had all the same ingredients except rox. All chickens received diet according to standard Poultry Research Centre diet composition (Table 1) - starter diet (3068 kcal kg−1; 23% crude protein) for two weeks, grower diet (3152 kcal kg−1; 20% crude protein) from two to four weeks, and finisher diet (3196 kcal kg−1; 19% crude protein) from four to five weeks of age. Rox was added to the starter and grower diets only. Chickens had ad libitum access to both feed and water (b1 μg As L−1).

2.2. Poultry litter sampling and analysis Samples of poultry litter (excreta mixed bedding material) were collected from each pen on 14, 28 and 35th days using a steel core of 10 cm diameter. Fifteen fresh samples were collected randomly up to the full depth of litter, and then total amount of litter in each pen was estimated by extrapolating the measurements to the whole pen area. These 15 litter samples, on each respective sampling day, were pooled together and a representative sample (a quarter of the composite sample; ~0.5 kg) was put into a double Ziplock® polyethylene bag, sealed and frozen at −20 °C until chemical analysis was performed. The remaining sample was put back into the pen and mixed well with the litter. The total litter amount was also determined at 35th day (clean out day) by both estimating through sampling method employed for day 14, 28 and 35, and physically weighing all the litter collected from each pen. Total litter weight measured at 35th day was higher than the weight estimated through sampling method. Therefore, a factor of 1.4 was used, which represented an average ratio of measured to estimated poultry litter weight from 16 pens, to calculate the weight of poultry litter produced Table 1 Composition of different growth stage poultry feeds used in the experiment to feed chickens for 35 days. Ingredient (%)

Control

Rox

Startera Growerb Finisherc Starter Grower Finisher Corn, yellow grain Fat, vegetable Fish meal, menhaden Soybean meal Wheat, hard grain Calcium carbonate Dicalcium phosphate Sodium chloride L-Lysine

18 3.8 3.0 26.9 43 1.5 1.5 0.43 0.23

18 3.4 5.0 16.2 53 1.0 1.0 0.34 0.15

15 4.1 3.5 15.1 58 1.0 1.1 0.36 0.15

18 3.8 3.0 26.9 43 1.5 1.5 0.43 0.23

18 3.4 5.0 16.2 53 1.0 1.0 0.34 0.15

15 4.1 3.5 15.1 58 1.0 1.1 0.36 0.15

DL-Methionine

0.23

0.10

0.09

0.23

0.10

0.09

L-Threonine

0.05

0.10

0.03

0.05

0.10

0.03

Broiler vitamin premix (0.5% inclusion) Choline chloride premix (0.5% inclusion) Vitamin E 5000 IU kg−1 Generic enzyme (0.5% inclusion) Coccidiostat (Amprol) Growth promoter (Roxarsone)

0.50

0.50

0.50

0.50

0.50

0.50

0.50

0.50

0.50

0.50

0.50

0.50

0.30 0.05

0.30 0.05

0.30 0.05

0.30 0.05

0.30 0.05

0.30 0.05

0.05 0.000

0.05 0.000

0.05 0.000

0.05 0.005

0.05 0.005

0.05 0.000

a b c

0–14 days. 15–28 days. 29–35 days.

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in each pen (Table 2). The loss of litter due to periodical sampling over the study period was also accounted for during the final calculations. For As analysis, poultry litter samples were homogenized in an allplastic blender. Moist samples, equivalent to 1 g oven-dried sample, were used for digestion to avoid any As loss through volatilization during drying. However, the results were reported on dry-weight basis after using moisture correction factor for each litter sample used in the digestion. Litter samples were digested with concentrated nitric acid (HNO3) (A509P212, trace metal grade, Fisher Scientific) in a microwave oven (Ethos Sel, Milestone) using a modified USEPA method (USEPA, 1996), wherein temperature was ramped from room temperature to 180 °C within 10 min and maintained at 180 °C for 10 more minutes to facilitate the complete digestion of samples. After digestion, the vials were allowed to cool. The solution was diluted to 100 ml using nanopure water (Barnstead nanopure, Thermo Scientific), and filtered through 0.45 μm PTFE syringe filter (033911C, Fisher Scientific). One milliliter of filtered solution was taken in a 50 ml centrifuge tube and diluted to a final volume of 50 ml using 1% HNO3 solution. This acidification of samples was done to prevent any precipitation before ICP-MS (Inductively Coupled Plasma Mass Spectrometer) analysis (Javed et al., 2013). Approximately 10 ml of this prepared solution was transferred to a sampling tube and placed on the auto-sampler of ICP-MS (iCAP QThermo Scientific). During the analysis of experimental samples, continuous calibration blanks (CCB) to check any metal carry over, and continuous calibration verification (CCV) external standards (2 and 20 μg As L−1) were also included after every 10 samples to ensure the validity of the initial calibration of the instrument. External standards were prepared in 1% HNO3 using a stock standard (CLMS-2AN; SPEX Certi Prep). Scandium (Sc) and yttrium (Y) were added to the samples as internal standards for quantitation and accounting for any instrumental drift over time. The reagent and experimental blanks were also analyzed along with the experimental samples. The As concentration was lower than the limits of detection (LOD: 0.01 μg As L−1 determined using 3× standard deviation (σ) of intensities of 7 blanks + average of the 7 blank intensities) in all reagents and experimental blanks. All the CCB showed As concentrations lower than the LOD, and As

concentrations in all the CCVs were within ±10% of the actual values of As (2 and 20 μg L−1) (Javed and Siddique, 2016). 2.3. Mass balance calculations For mass balance calculations, the following equations were used to quantify As intake and As excreted in the poultry litter: I ¼ Wf Cf

ð1Þ

I = Cumulative As taken in by chickens through feed consumption (mg As pen−1). Wf = Amount of feed consumed by chicken (kg pen−1). Cf = Concentration of As in poultry feed (e.g. 14.24 mg As kg−1 rox feed). E ¼ ðWl Cl Þ

ð2Þ

E = Cumulative As excreted by chickens in poultry litter (mg As pen−1). Wl = Total amount of poultry litter producedby chickens (kg pen−1). Cl = Concentration of As in poultry litter (mg kg−1). Recovery of As ð%Þ ¼ E=I 100

ð3Þ

3. Results 3.1. Poultry production with feed consumption Data pertaining to number of chickens, cumulative feed consumption and poultry litter production per pen, and average body weight per chicken on different days of observation have been given in Table 2. The experiment was started with 102 chickens in each pen but their number decreased with time due to death and removal for meat analyses. By 35th day, the numbers of Cobb chickens remained were 69 ± 5 and 67 ± 5 pen−1 in the control and rox treatments,

Table 2 Measurements of different growth parameters and poultry litter production used in the mass balance calculations to determine the fate of feed additive roxarsone. Measured characteristics

−1

Number of chickens pen

Average body weight of chicken (kg chicken−1)

Cumulative feed consumption (kg pen−1)

Estimated weight of poultry litter produced (kg pen−1)a

Calculated weight of poultry litter produced (kg pen−1)b

Strain

Cobb Cobb Ross Ross Cobb Cobb Ross Ross Cobb Cobb Ross Ross Cobb Cobb Ross Ross Cobb Cobb Ross Ross

Treatment

Control Rox Control Rox Control Rox Control Rox Control Rox Control Rox Control Rox Control Rox Control Rox Control Rox

(Days) 0

14

28

35

102 ± 0.6 102 ± 0.6 102 ± 0.0 102 ± 0.6 0.043 ± 0.0003 0.040 ± 0.004 0.042 ± 0.003 0.040 ± 0.0002

88 ± 1.5 88 ± 0.6 88 ± 1.5 89 ± 0.6 0.40 ± 0.005 0.41 ± 0.007 0.40 ± 0.022 0.41 ± 0.005 47 ± 1 46 ± 1 47 ± 2 48 ± 1 30 ± 4 30 ± 2 30 ± 6 29 ± 3 43 ± 6 42 ± 3 41 ± 9 41 ± 4

81 ± 5.3 80 ± 5.3 83 ± 1.2 84 ± 0.6 1.55 ± 0.02 1.55 ± 0.06 1.53 ± 0.05 1.58 ± 0.03 193 ± 6 187 ± 9 186 ± 5 192 ± 7 57 ± 4 52 ± 2 54 ± 3 53 ± 1 80 ± 5 73 ± 3 76 ± 4 74 ± 17

69 ± 5.5 67 ± 4.7 71 ± 2.5 72 ± 1.5 2.20 ± 0.03 2.25 ± 0.02 2.17 ± 0.09 2.21 ± 0.03 297 ± 20 282 ± 14 300 ± 41 292 ± 12 59 ± 3 59 ± 1 59 ± 9 59 ± 3 82 ± 4 83 ± 2 82 ± 12 83 ± 4

19 ± 2 18 ± 4 20 ± 3 19 ± 5 19 ± 2 18 ± 4 20 ± 3 19 ± 5

Values are the means of the three replications with associated standard deviations (±). a Initially (day 0) a known weight of wood chips was spread on each pen floor. Afterwards on 14th, 28th and 35th days, the weight of poultry litter was estimated based on sample weight and pen area. b On 35th day, litter in each pen was also weighed physically. After comparing the estimated and measured weights on 35th day, a factor of 1.4 (average ratio of measured to estimated poultry litter weight from 16 pens) was used to calculate the poultry litter produced. These values were used in the mass balance to calculate the amount of arsenic excreted by chicken in each pen.


305

respectively, while 71 ± 3 and 72 ± 2 Ross chickens pen−1 remained in each control and rox treated pens, respectively. Cumulative feed consumption increased significantly with age for both the strains under both the treatments. In 35 days, Cobb chickens consumed 297 ± 20 and 282 ± 14 kg feed pen−1 under the control and rox treatment, respectively. Similar feed consumption 300 ± 41 and 292 ± 12 kg pen−1 was recorded in control and rox fed pens, respectively, growing the Ross strain. The average body weight of chickens also increased with age for both the strains, and no significant differences were observed within the chicken strains and the two treatments. The average body weight ranged between 2.17 ± 0.09 to 2.25 ± 0.02 kg chicken−1 at day 35. 3.2. Arsenic intake Total As intake was calculated using the amount of feed consumed and concentration of As present in the feed (Eq. (1)). In control feed, rox was not added (Table 1), therefore we assumed that no As would be taken up by the chickens under the control treatment. In the rox treatment, As was added at 14.24 mg kg−1 of feed. As the chickens grew over time, they consumed more feed, and consequently, more As was taken up by chickens while consuming rox-treated starter diet (0–14 days) and grower diet (14–28 days). Supply of As was stopped after 28th day as the grower diet was replaced by the finisher diet which did not contain rox. Because the two strains were not different in their feed consumption (Table 2), As intake calculated for both strains was similar. At 14th day, As intake was 661 ± 10 and 680 ± 8 mg pen−1 which increased cumulatively to 2669 ± 128 and 2730 ± 92 mg pen−1 in Cobb and Ross, respectively at 28th day (Table 3). 3.3. Poultry litter production and arsenic concentration in the poultry litter Initially, a known amount of wood chips was spread on each pen floor. As the chickens grew in age and size, they consumed feed and water, and excreted urine and feces on to the bedding material. Consequently, weight of poultry litter (mixture of wood chips and excreta) increased significantly with time. Cobb chickens produced 43 ± 6 and 42 ± 3 kg dry poultry litter pen−1 at 14th day and 80 ± 5 and 73 ± 3 kg litter pen−1, in control and rox-fed pens, respectively at 28th day. Similarly, the poultry litter production by Ross increased from 41 ± 9 and 41 ± 4 kg litter pen−1 at 14th day to 76 ± 4 and 74 ± 17 kg litter pen−1 at 28th day under control and rox treatments, respectively (Table 2). Though higher amounts of poultry feed were consumed during the last week (29–35 days) of chicken growth, not much corresponding increase in poultry litter was recorded at 35th day that obviously indicates some discrepancy in poultry litter determination at 35th day. Regarding As concentrations in poultry litter collected from control pens, 0.25 ± 0.1 to 1.75 ± 0.4 mg kg−1 As was detected in the litter from both the strains analyzed during their growth period (Fig. 1) even though the control feed was devoid of rox. Arsenic concentration was significantly higher in the poultry litter of rox-fed chickens than

Fig. 1. Arsenic concentration in poultry litter (A) produced by two strains of chicken (Cobb and Ross) and cumulative arsenic excreted by both the strains (B). Cobb-cont and Rosscont are chickens that received poultry feed without roxarsone, whereas Cobb-Rox and Ross-Rox received poultry feed containing roxarsone. Error bars showed ± standard deviation from 3 replications.

the poultry litter produced in the control treatment. With time, the concentration of As increased significantly in the poultry litter collected under both the strains. Mean As concentration was 17 ± 3 and 15 ± 4 mg kg−1 when measured at 14th day and 40 ± 7 and 36 ± 4 mg kg−1 at 28th day in the poultry litter of Cobb and Ross, respectively, which decreased to 23 ± 2 and 24 ± 2 mg kg−1, respectively at 35th day as the grower diet was replaced by the finisher diet after 28th day which did not contain rox (Fig. 1A).

Table 3 Mass balance of arsenic taken up by chicken fed with roxarsone containing feed and arsenic excreted in the chicken poultry litter. Parameters

Strain

Day 14

Day 28

Day 35

Arsenic intake (mg pen−1)

Cobb Ross Cobb Ross Cobb Ross

661 (±10) 680 (±8) 703 (±149) 625 (±185) 106 ± 21 92 ± 26

2669 (±128) 2730 (±92) 2896 (±414) 2362 (±228) 108 ± 11 86 ± 5

1838 (±192) 2109 (±94) 69 ± 4 77 ± 4

Arsenic excreted (mg pen−1) Arsenic recovered in poultry litter (%)

Arsenic recovery for day 35 was calculated using arsenic intake data calculated for day 28 because arsenic containing rox feed was stopped at day 28. Parenthesis shows ± standard deviation calculated from three replications.

306


3.4. Mass balance to determine recovery of As in the poultry litter Excretion of As in litter was calculated using Eq. (2). At 14th day, As excretions by Cobb and Ross were 703 ± 149 and 625 ± 185 mg pen−1, which increased to 2896 ± 414 and 2362 ± 228 mg pen−1, respectively, by 28th day (Table 3). At 35th day, 1838 ± 192 and 2109 ± 94 mg As pen−1 was determined in Cobb and Ross pens as excreted As (Table 3, Fig. 1). Small amount of As (101 ± 21 to 141 ± 29 mg pen−1) was also excreted in the control treatment (Fig. 1B). Mass balance calculation (Eq. (3)) was performed to determine the recovery of As in the poultry litter produced during the experiment (Table 3). During the first 14 days, 106 ± 21% and 92 ± 26% of ingested As was recovered from the poultry litter produced by Cobb and Ross strains, respectively. Similar recovery of As (86 ± 5 to 108 ± 11%) was achieved at 28th day when feeding chicken with rox containing grower diet was stopped and cumulative As excreted in poultry litters produced by Cobb and Ross was determined. Arsenic recovery decreased in the poultry litters of Cobb and Ross when calculated for 35th day due to decreased As concentration in the poultry litter and some discrepancy in poultry litter measurements at 35th day (Table 3). 4. Discussion The mass balance of As from poultry feed to poultry litter reported in this manuscript is a part of a comprehensive study envisaged to track As from rox containing poultry feed to poultry litter including its metabolism in the chicken meat (Peng et al., 2014; Liu et al., 2016; Peng et al., 2017). Two strains of chickens (Cobb and Ross) were grown on feed with or without the addition of rox in separate pens. Quite expectedly, cumulative feed consumption and body weight of the chickens increased with age. Intake of As by the chickens kept increasing with time in rox treatment as starter and grower diets contained recommended dose of rox. The concentration of As in the poultry litter of rox treatment increased till 28th day and then decreased when poultry litter was analyzed at 35th day. Because as the chickens grew in size and weight between 28th and 35th day, they consumed rox-free (Finisher) diet and produced excreta, which led to the decrease in As concentration when As was measured in litter at 35th day (Fig. 1A). Arsenic concentration of 0.25–1.75 mg kg−1 was also detected in the litters of control treatment chickens that indicated the presence of As in the poultry feed even without the addition of rox. Previously, a similar study also reported 0.6 mg As kg−1 poultry litter where chickens were not fed with rox (Garbarino et al., 2003). In our study, the source of As in the control feed was menhaden fishmeal, a protein source in the feed (Table 1) (Liu et al., 2016). Though the concentrations of As in poultry litter reported in previous studies vary significantly from 0 to 77 mg kg−1, there is no mention of recovery or mass balance of As from poultry feed to poultry litter (Morrison, 1969; Sims and Wolf, 1994; Anderson and Chamblee, 2001; Arai et al., 2003; Toor et al., 2007; Fisher et al., 2015). For mass balance calculation to determine the recovery of As in poultry litter, we used As intake calculated for 14th and 28th days and compared with the As excreted at both the time points (Table 3). The results (86–108% As recovery in poultry litter at 14th and 28th days) suggested that there was no significant retention of As in the body of chickens fed on rox containing feed. These mass balance results are corroborated by the results of As in chicken meat which revealed insignificant retention of As in the meat of chickens sampled from the pens (in the current experiment) on 35th day and taken for the analyses of different As species (Peng et al., 2014; Liu et al., 2016; Peng et al., 2017; Liu et al., 2018). Breast meat from the control and rox-fed chickens showed very minute As concentrations; meat from rox-fed treatment contained ~42 μg kg−1 (rox plus all individual As species) versus ~33 μg kg−1 in the control treatment chicken breast meat (Liu et al., 2016). The liver samples from these chickens accumulated relatively higher concentrations of rox and other individual As species (~10 times higher than the breast meat); ~400 μg kg−1 in rox-fed chicken compared to ~80 μg kg−1 in

the control chicken (Peng et al., 2014). If we assume and apply this liver As concentration to whole chicken body, even then it would not contribute much (0.4 mg kg−1) to our overall mass balance calculations that deals higher As concentrations in poultry feed and litter, and the meat As concentrations are far below the tolerable levels (2.0 mg kg−1 previously established by FDA before 1963) (Nachman et al., 2013). Therefore, our results do not align with the findings of FDA (2011) where higher concentrations of total As (2.8 ± 1.4 mg kg−1) in liver samples of rox-fed chicken were reported. However, recent findings on the identification of additional three methylated phenylarsenical metabolites in the liver of chickens fed with rox containing feed from this study revealed that the concentrations of metabolites (~500 μg kg−1) in liver decreased to ~130 μg kg−1 after 5 days of rox feed cessation; these residual As species in chicken liver are relevant to human exposure if chicken livers are consumed (Peng et al., 2017). Arsenic recovery for 35th days was also determined by comparing cumulative As intake at 28th day when chickens stopped receiving rox containing feed, with excreted As at 35th day. A recovery of 69–77% was calculated in both Cobb and Ross chicken strains. This lower As recovery is attributed to discrepancy in poultry litter determination at 35th day and/or other factors such as environmental losses due volatilization, or loss of As to the pen floor via urine (Nigra et al., 2017). This study also revealed that the poultry litter from rox-fed chickens contained higher As (~40 mg kg−1). Because poultry litter is commonly used as an organic amendment to fertilize soil (Wilkinson, 1979) for crop production, As and its different metabolites/species can be translocated to different plant parts that may arise some public health issues (Rutherford et al., 2003; Yao et al., 2009; Yao et al., 2016). 5. Conclusions The results of our study show that arsenic from poultry feed ends up in poultry litter when two poultry stains are fed with rox-amended feed. Our mass balance calculations showed As recovery between 86 and 108% during the growth period of 28 days which suggested no significant retention of As in the chickens. Our complementary studies on chicken meat analysis also substantiated our mass balance results. Higher arsenic in poultry litter warrants investigations on its environmental implications if poultry litter enriched in As is applied to agricultural soils for crop production. Acknowledgements Authors gratefully acknowledge the financial support provided by Natural Sciences and Engineering Research Council of Canada (NSERC DG; 371909-2009), Canada Foundation for Innovation (128377), Canadian Water Network, Poultry Industry Council, and Alberta Livestock and Meat Agency Ltd. (ALMA). References Anderson, B.K., Chamblee, T.N., 2001. The effect of dietary 3-nitro-4-hydroxyphenyl arsenic acid (roxarsone) on the total arsenic level in broiler excreta and broiler litter. J. Appl. Poult. Res. 10, 323–328. Arai, Y., Lanzirotti, A., Sutton, S., Davis, J.A., Sparks, D.L., 2003. Arsenic speciation and reactivity in poultry litter. Environ. Sci. Technol. 37, 4083–4090. ATSDR (Agency for Toxic Substances and Disease Registry), 2007. Toxicological Profile for Arsenic. U.S. Department of Health and Human Service https://www.atsdr.cdc.gov/ toxprofiles/tp2.pdf, Accessed date: 5 February 2018. Aviagen, 2009. Ross Broiler Nutrition Supplement. Aviagen, Huntsville, AL, USA. Cobb-Vantress, 2008. Cobb Broiler Management Guide. Cobb-Vantress, Siloam Springs, AR, USA. European Commission, 1999. On the Undesirable Substances and Products in Animal Nutrition. Council Directive 1999/29/EC. http://eur-lex.europa.eu/LexUriServ/LexUriServ. do?uri=CONSLEG:1999L0029:19990504:EN:PDF accessed Dec 31, 2017. FDA (Food and Drug Administration), 1963. Food additives resulting from contact with containers or equipment and food additives otherwise affecting food. Fed. Reg. 28, 10871. FDA, 2011. Final Report on Study 275.30. Provide Data on Various Arsenic Species Present in Broilers Treated With Roxarsone: Comparison With Untreated Birds. https://www. fda.gov/downloads/AnimalVeterinary/SafetyHealth/ProductSafetyInformation/ UCM257545.pdf, Accessed date: 31 December 2017.

S.K. Gupta et al. / Science of the Total Environment 630 (2018) 302–307 FDA, 2013. FDA's Response to the Citizen Petition (FDA-2009-p-0594). http://www. centerforfoodsafety.org/files/20130930_docket-fda-2009-p-0594_signed-arsenic-cpresponse_94793.pdf, Accessed date: 5 February 2018. Fisher, D.J., Yonkos, L.T., Staver, K.W., 2015. Environmental concerns of roxarsone in broiler poultry feed and litter in Maryland, USA. Environ. Sci. Technol. 49, 1999–2012. Garbarino, J.R., Bednar, A.J., Rutherford, D.W., Beyer, R.S., Wershaw, R.L., 2003. Environmental fate of roxarsone in poultry litter. 1. Degradation of roxarsone during composting. Environ. Sci. Technol. 37, 1509–1514. Javed, M.B., Siddique, T., 2016. Thermally released arsenic in porewater from sediments in the Cold Lake area of Alberta, Canada. Environ. Sci. Technol. 50, 2191–2199. Javed, M.B., Kachanoski, G., Siddique, T., 2013. A modified sequential extraction method for arsenic fractionation in sediments. Anal. Chim. Acta 787, 102–110. Liu, Q., Peng, H., Lu, X., Zuidhof, M.J., Li, X., Le, X.C., 2016. Arsenic species in chicken breast: temporal variations of metabolites, elimination kinetics and residual concentrations. Environ. Health Perspect. 124, 1174–1181. Liu, Q., Lu, X., Peng, H., Popowich, A., Tao, J., Uppal, J.S., Yan, X., Boe, D., Le, X.C., 2018. Speciation of arsenic – a review of phenylarsenicals and related arsenic metabolites. Trends Anal. Chem. https://doi.org/10.1016/j.trac.2017.10.006. Moe, B., Peng, H.Y., Lu, X.F., Chen, B.W., Chen, L.W.L., Gabos, S., Li, X.-F., Le, X.C., 2016. Comparative cytotoxicity of fourteen trivalent and pentavalent arsenic species determined using real-time cell sensing. J. Environ. Sci. 49, 113–124. Morrison, J.L., 1969. Distribution of arsenic from poultry litter in broiler chickens, soil and crops. J. Agric. Food Chem. 17, 1288–1290. Nachman, K.E., Raber, G., Francesconi, K.A., Navas-Acien, A., Love, D.C., 2012. Arsenic species in poultry feather meal. Sci. Total Environ. 417–418, 183–188. Nachman, K.E., Baron, P.A., Raber, G., Francesconi, K.A., Navas-Acien, A., Love, D.C., 2013. Roxarsone, inorganic arsenic, and other arsenic species in chicken: a US-based market basket sample. Environ. Health Perspect. 121, 818–824. Naujokas, M.F., Anderson, B., Ahsan, H., Aposhian, H.V., Graziano, J.H., Thompson, C., Suk, W.A., 2013. The broad scope of health effects from chronic arsenic exposure: update on a worldwide public health problem. Environ. Health Perspect. 121, 295–302. Nigra, A.E., Sanchez, T.R., Nachman, K.E., Harvey, D.E., Chillrud, S.N., Graziano, J.H., NavasAcien, A., 2017. The effect of the Environmental Protection Agency maximum contaminant level on arsenic exposure in USA from 2003 to 2014: an analysis of the

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National Health and Nutrition Examination Survey (NHANES). Lancet Public Health 2:e513–21. https://doi.org/10.1016/s2468-2667(17)30195-0 (accessed on January 22, 2018). Peng, H., Hu, B., Liu, Q., Yang, Z., Lu, X., Huang, R., Li, X., Zuidhof, M.J., Le, X.C., 2014. Liquid chromatography combined with atomic and molecular mass spectrometry for speciation of arsenic in chicken liver. J. Chromatogr. A 1370, 40–49. Peng, H., Hu, B., Liu, Q., Li, J., Li, X.-F., Zhang, H., Le, X.C., 2017. Methylated phenylarsenicals metabolites discovered in chicken liver. Angew. Chem. Int. Ed. 56, 6773–6777. Rutherford, D.W., Bednar, A.J., Garbarino, J.R., Needham, R., Staver, K.M., Wershaw, R.L., 2003. Environmental fate of roxarsone in poultry litter. Part II. Mobility of arsenic in soils amended with poultry litter. Environ. Sci. Technol. 37, 1515–1520. Schmidt, C.W., 2012. Maryland bans arsenical drug in chicken feed. Environ. Health Perspect. 120, a269. Sims, J.T., Wolf, D.C., 1994. Poultry waste management - agricultural and environmental issues. Adv. Agron. 52, 1–83. Toor, G.S., Haggard, B.E., Donoghue, A.M., 2007. Water extractable trace elements in poultry litters and granulated products. J. Appl. Poult. Res. 16, 351–360. USEPA (U. S. Environmental Protection Agency), 1996. Microwave Assisted Acid Digestion of Siliceous and Organically Based Matrices. Method 3052. http://www.epa.gov/sites/ production/files/2015-12/documents/3052.pdf, Accessed date: 31 December 2017. Wilkinson, S.R., 1979. Plant nutrient and economic value of animal manures. J. Anim. Sci. 48, 121–133. Yang, Z., Peng, H., Lu, X., Liu, Q., Huang, R., Hu, B., Kachanoski, G., Zuidhof, M.J., Le, X.C., 2016. Arsenic metabolites, including N-acetyl-4-hydroxy-m-arsanilic acid, in chicken litter from a roxarsone-feeding study involving 1600 chickens. Environ. Sci. Technol. 50, 6737–6743. Yao, L.X., Li, G.L., Dang, Z., He, Z.H., Zhou, C.M., Yang, B.M., 2009. Arsenic speciation in turnip as affected by application of chicken manure bearing roxarsone and its metabolites. Plant Soil 316, 117–124. Yao, L., Huang, L., He, Z., Zhou, C., Lu, W., Bai, C., 2016. Delivery of roxarsone via chicken diet to chicken to chicken manure to soil to rice plant. Sci. Total Environ. 566-567, 1152–1158.