Occurrence and Profiles of Melamine and Cyanuric Acid in Bovine

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Ecotoxicology and Human Environmental Health

Occurrence and Profiles of Melamine and Cyanuric Acid in Bovine Feed and Urine from China, India, and the United States Hongkai Zhu, Bommanna G. Loganathan, and Kurunthachalam Kannan Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.9b00469 • Publication Date (Web): 07 May 2019 Downloaded from http://pubs.acs.org on May 8, 2019

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Environmental Science & Technology

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Occurrence and Profiles of Melamine and Cyanuric Acid in Bovine Feed and Urine

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from China, India, and the United States

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Hongkai Zhu,† Bommanna G. Loganathan,§ and Kurunthachalam Kannan*†#

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†Wadsworth

Center, New York State Department of Health, and Department of Environmental

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Health Sciences, School of Public Health, State University of New York at Albany, Empire State

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Plaza, P.O. Box 509, Albany, New York 12201-0509, United States

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§Department

of Chemistry and Watershed Studies Institute, Murray State University, 1201 Jesse

D. Jones Hall, Murray, Kentucky 42071-3300, United States #Biochemistry

Department, Faculty of Science and Experimental Biochemistry Unit, King Fahd

Medical Research Center, King Abdulaziz University, Jeddah 21589, Saudi Arabia

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*Corresponding author at: Wadsworth Center, Empire State Plaza, P.O. Box 509, Albany, New

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York 12201-0509, United States

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Fax: +1 518 473 2895

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E-mail: [email protected] (K. Kannan)

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Submitted to: Environmental Science & Technology

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1 ACS Paragon Plus Environment


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Graphic Abstract

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■ ABSTRACT

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Melamine and cyanuric acid have been reported to occur in animal products. Nevertheless,

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information that pertains to the occurrence of melamine and cyanuric acid in cattle feed and urine

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is lacking. In this study, the occurrence of melamine and its three derivatives (i.e., cyanuric acid,

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ammeline, and ammelide) was determined in 183 bovine urine and 29 matched feed samples

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collected from China, India, and the United States. ∑Melamine (sum of four target compounds)

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was found in all urine samples at concentrations that ranged from 4.2 to 5280 ng/mL (median: 370

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ng/mL); cyanuric acid was the major derivative, accounting for 97% of the total concentrations,

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followed by melamine (2.2%). The ubiquitous occurrence of ∑Melamine in feed (21–6230 ng/g)

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suggests that it is the major source of melamine and its derivatives in bovines. Urinary

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concentrations of melamine and cyanuric acid varied significantly among the three countries, with

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samples from China as having the highest concentrations, followed by the United States and India.

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The calculated cumulative daily intakes of melamine and cyanuric acid were at least 10-fold below

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the current tolerable daily intake recommended for humans. Our study provides evidence-based

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data on exposure patterns and sources of melamine and cyanuric acid in cattle.

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■ INTRODUCTION

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Melamine has received considerable public attention due to food scandals, such as pet food

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contamination in North America in 2007 and the toxic milk powder scandal in China in 2008.1-3

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Melamine was illegally added to food products to raise the protein content, which resulted in

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devastating human or animal health consequences in both cases. Since then, several countries

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around the globe have established food (infant formula, milk, and dietary products)4-7 and

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environmental (indoor dust, air, soil, sewage sludge, and sediment)8-12 surveillance programs. In

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2010, melamine was recognized as an emerging contaminant.13

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Melamine was widely detected in urine samples collected from the general United States

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population in recent years, with the measured concentrations in the range of below the limit of

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detection to 1,090 ng/mL.14-16 This highlights ongoing melamine exposure even after a decade of

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the above-noted food scandals. A clinicopathological study indicated that a urinary melamine

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concentration above 7.1 μg/mmol creatinine was suggestive of acute exposures.17 Several

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laboratory animal exposure studies have suggested nephrotoxic effects of melamine at chronic

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low-dose exposures.18-21 A growing body of evidence also suggests neurotoxicity, reproductive

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toxicity, and genotoxicity of melamine.22-25 In addition, an epidemiologic study showed interactive

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effect of melamine with di-(2-ethylhexyl) phthalate in exacerbation of renal damage in children.26

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Nevertheless, little is known about the sources and pathways of exposure to melamine and cyanuric

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acid in farm animals, as their products (meat/dairy) are sources of human exposure.

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In a recent study, we found melamine in infant formula and dairy products purchased in the

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United States market in 2018.7 Environmental sources have been suggested to contribute to the

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continued occurrence of melamine in dairy products. Melamine is also widely used in kitchenware

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and tableware.27, 28 Food chain transfer of melamine through farm products (e.g., dairy, meat) is


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an important source of human exposure.8, 29-33 Nevertheless, little is known with regard to the

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occurrence and food chain transfer of melamine and its derivatives in farm animals. Further,

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derivatives of melamine, such as cyanuric acid, have received little or no attention in previous

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human exposure studies.

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Urine is a suitable matrix for the assessment of melamine exposure, as over 90% of melamine

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is excreted unchanged in urine.34, 35 Whereas studies have reported the occurrence of melamine

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and cyanuric acid in urine from humans and pet dogs and cats,16, 36 little is known about melamine

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exposure in bovines. Cyanuric acid has been approved for use in cattle feed as a non-protein

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nitrogen source in the United States.37 Nevertheless, no earlier studies have determined the

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occurrence of melamine and cyanuric acid in cattle feed. The transfer of melamine and cyanuric

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acid from feed to farm products, especially dairy products, merits investigation.

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In this study, bovine feed and urine samples were collected from China, India and the United

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States to (1) elucidate the occurrence and profiles of melamine, cyanuric acid, ammeline, and

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ammelide; (2) examine geographic patterns in distribution of melamine and its derivatives in

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bovine urine and feed; and (3) estimate exposure doses of melamine and cyanuric acid in bovines

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from measured urinary concentrations. These results were extrapolated to further understand the

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carryover of melamine and cyanuric acid from feed to milk.

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■ MATERIALS AND METHODS

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Sample Collection. A total of 183 bovine urine samples were collected from cattle farms in

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Northern China (n = 100); Mettupalayam, India (n = 45); and Murray, Kentucky, United States (n

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= 38) during the period of March through November 2018. These urine samples were obtained

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from nine different breeds, i.e., two from China (Simmental and Holstein), three from India (Jersey,

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Indian Buffalo, and an indigenous breed), and four from the United States (Angus, Corriente, 5 ACS Paragon Plus Environment


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Shorthorn, and Jersey). There were 60 males (33%) and 123 females (67%), with an age range of

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4–240 months. The bovines were further grouped as dairy cows (n = 110; 60%), beef cattle (n =

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58; 32%), and buffalos (n = 15; 8%; all from India). Further details regarding the samples are given

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in the Supporting Information (SI; Tables S1). Cow/buffalo urine was collected directly in a wide-

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mouthed polypropylene (PP) container and subsequently transferred into a new PP tube. Both PP

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containers and tubes were cleaned in methanol prior to use. To further explore the sources of target

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compounds present in bovine urine, feed samples were provided by the cattle farms where the

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urine samples were collected. In total, 29 feed samples were collected from China (n = 16), India

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(n = 7), and the United States (n = 6). Several types of feed were obtained, including grains,

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grasses/hay, feed additives, premixed formula, and concentrated formula, which are commonly

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fed to the bovines. The cattle also were allowed graze in open pastures/grasslands, especially in

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India and the United States. Further details of the feed samples are provided in Table S2.

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Cow/buffalo urine and feed samples were extracted immediately and stored at -20° C at

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Wadsworth Center, New York State Department of Health, for instrumental analysis.

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Chemicals and Reagents. Native standards of melamine (99% purity), ammeline (97.9%),

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ammelide (98%), and cyanuric acid (98%) were purchased from Sigma-Aldrich (St. Louis, MO).

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Corresponding isotopically labeled internal standards (ISs), namely 15N3,13C3-melamine (15N3 at

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98%,

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cyanuric acid (15N3 at 98%, 13C3 at 99%) were purchased from Cambridge Isotope Laboratories

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(Andover, MA). Ammonium formate (> 99.995% purity) and ammonium hydroxide (28.0–30.0%

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NH3 basis; NH4OH) were purchased from Sigma-Aldrich. High-performance liquid

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chromatography (HPLC)-grade acetonitrile, water, ethyl acetate, and reagent-grade formic acid

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(FA) were purchased from J. T. Baker (Phillipsburg, NJ). HPLC-grade isopropanol was purchased

13C

3

at 99%),

13C

3-ammeline

(13C3 at 99%),

13C

3-ammelide


(13C3 at 99%), and

15N ,13C 3 3

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from Honeywell B&J (Morristown, NJ). Oasis MAX and Oasis MCX solid phase extraction (SPE)

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cartridges (150 mg, 6 mL, 30 μm particles) were purchased from Waters Corporation (Milford,

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MA).

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Analytical Methods. Details of the sample pre-treatment and instrumental analysis have been

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reported in our previous studies10, 35 and are provided in the SI. Briefly, cow/buffalo urine samples

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were extracted with a mixture of ethyl acetate/isopropanol (95:5, v/v). Cow feed samples were

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extracted with methanol and purified, using a mixed-mode SPE method. Because the polarity of

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cyanuric acid is different from that of the other three target compounds, two separate sample

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preparation procedures were used. One aliquot was acidified with FA for cyanuric acid analysis;

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the other aliquot was basified with NH4OH for the analysis of melamine, ammeline, and ammelide.

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Identification and quantification of melamine and its derivatives were performed on a Shimadzu

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LC-30AD Series HPLC system, interfaced with ABSciex 5500 triple-quadrupole mass

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spectrometry (MS/MS). Analytes were separated using acetonitrile (A) and 5 mM ammonium

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formate buffer (pH = 4.0; B) by passing them through a Luna hydrophilic-lipophilic interaction

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liquid chromatography column (100 mm × 3.0 mm, 3.0 μm particle size; Phenomenex, Torrance,

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CA). Two acquisition modes were used; i.e., the negative mode was applied for cyanuric acid and

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ammelide detection (0–4.2 min), and the positive mode was applied for melamine and ammeline

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detection (4.2–18 min). The multiple reaction monitoring mode was used for the identification of

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target compounds; quantitative and qualitative m/z ion transitions of target compounds are listed

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in Table S3. Typical chromatograms of standard and real bovine urine and feed samples are shown

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in Figure S1. Creatinine (CR) was determined in cow/buffalo urine, using HPLC-MS/MS38 to

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account for urine dilution.



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Quality Assurance (QA)/Quality Control (QC). Blanks (field, procedural, and solvent),

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matrix spikes, and midpoint calibration standards were analyzed for monitoring background

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contamination, analytical method validation, and instrumental drift in sensitivity, respectively.

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Field and procedural blanks were prepared with HPLC-grade water (in lieu of urine) and passed

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through the entire analytical procedure. The HPLC syringe was rinsed with a mixture of

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acetonitrile/water (50:50, v/v) before and after any injection. Target compounds were not detected

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in field, procedural, or solvent (acetonitrile) blanks, which indicated no background level of

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contamination or carryover of target chemicals between injections. The recoveries of target

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compounds (fortified at two levels of 5 and 50 ng each) in spiked matrixes (n = two spiked level

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× triplicate × two matrixes = 12) ranged from 74% (cyanuric acid) to 96% (melamine) with a

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relative standard deviation of 0.99), was used in the

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quantification of the target analytes. The limits of quantification (LOQs), derived from the lowest

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point in the calibration standard with a signal-to-noise ratio of 10 and a nominal sample weight

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(for urine or feed), are summarized in Table 1. Concentrations of target chemicals in animal feed

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are presented on a products basis (as marketed; ng/g wet weight).

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Data Analysis. The concentrations below the LOQs were assigned one-half of that value for

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statistical analysis. Because the data were not normally distributed (Shapiro-Wilk test; p < 0.05),

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nonparametric statistical tests were applied to assess the statistical significance. A Mann-Whitney

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U test was used to compare differences in the concentrations of melamine and its derivatives

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among the three countries. Spearman’s correlation analysis was used to examine the relationship

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between melamine and its derivatives. Statistical analyses were performed with SPSS version 17.0


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software package. All of the plots were created with Origin version 8.1. The statistical significance

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was set at p < 0.05.

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■ RESULTS AND DISCUSSION

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Melamine and its Derivatives in Bovine Urine and Feed. The measured concentrations of

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melamine and its derivatives in bovine urine and feed samples are presented in Table 1 and Tables

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S1–S2. Melamine and cyanuric acid were found in all bovine urine samples (100%), whereas

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ammeline and ammelide were found in 3.8% and 50% of the samples, respectively. The sum

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concentrations (∑3melamine; sum of melamine, cyanuric acid and ammelide. Ammeline was

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excluded here due to its low detection frequency) varied by 3 orders of magnitude, ranging from

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4.2 to 5280 ng/mL, across the three countries. ∑3Melamine concentrations (median: 370 ng/mL)

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in bovine urine were significantly higher than those reported for pet dog/cat urine from the United

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States (94 ng/mL for dogs and 170 ng/mL for cats).36 Cyanuric acid was the predominant

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compound found in bovine urine, followed by melamine. The overall median concentrations of

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cyanuric acid (360 ng/mL) in bovine urine were approximately 100-fold higher than those of

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melamine (3.4 ng/mL). It is worth to note that cyanuric acid can be derived from the hydrolysis of

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melamine in animal bodies.1 Nevertheless, toxicokinetic studies have reported that melamine was

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largely (> 90%) excreted unmetabolized in urine within 24 h of exposure.14, 30 Therefore, high

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cyanuric acid concentrations found in bovine urine suggest direct exposure of this compound (as

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discussed below).

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To further confirm the sources of target chemicals in bovine urine, cattle feed samples collected

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from the three countries were analyzed. Melamine and its three derivatives were found in all feed

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samples, and ∑4melamine (sum of four target compounds) concentrations ranged from 21 to 6230



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ng/g (Table 1). The concentrations of cyanuric acid (median: 180 ng/g) in bovine feed were 20-

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fold higher than those of melamine (7.4 ng/g). These results further indicated that feed is the direct

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source of exposure to melamine and cyanuric acid in cows and buffalos. Cyanuric acid has been

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authorized for use as a component (up to 30%) in feed-grade biuret in the United States, whereas

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melamine was not allowed for use in animal feed.37 In 2007, melamine was illegally added to

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animal feed, which resulted in the deaths of hundreds of pet dogs and cats in North America. In

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comparison to those reported levels of up to 8.4% in pet foods,39 melamine concentrations

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measured in bovine feed in our study were several orders of magnitude lower (range: 1.3–170

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ng/g). These results indicate that melamine and cyanuric acid found in feed originated from

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environmental sources and migration of these compounds through soil-crop-feed pathway.

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Melamine and cyanuric acid are frequently added to certain fertilizers, from which they

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accumulate in soil and then are taken up by crops. Application of sewage sludge is another source

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of soil contamination by melamine.11 Other sources of melamine include cyromazine, a registered

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insecticide sprayed on various crops and as a larvicide incorporated into cattle feed.40 In both

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animals and plants, cyromazine can be metabolized to melamine, and then to ammeline, ammelide,

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and cyanuric acid.41

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Cyanuric acid accounted for 97% and 74% of ∑melamine concentrations in bovine urine and

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feed samples, respectively, which was followed by melamine at 2.2% in urine and 7.8% in feed

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(Figure 1). Ammeline and ammelide collectively accounted for 0.47% and 18% of the ∑melamine

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concentrations in urine and feed, respectively. Similar to that found in this study, cyanuric acid

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(83%) was the most abundant derivative found in infant formula purchased in the United States in

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2018.7 The proportion of melamine (16–44%) in human and pet urine samples, however, was

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higher than that found in bovine urine.15, 16, 36 A likely explanation for this pattern of melamine


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and cyanuric acid is exposure sources. Melamine is widely used in household products, including

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furniture, dinnerware, and food utensils.42 A higher proportion of melamine to ∑melamine

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concentration was found in indoor dust (57%), dairy products (24%), and sludge (46%). Thus, a

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relatively higher proportion of melamine in human and pet urine samples than that in bovine urine

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suggests exposure from indoor sources that contain high concentrations of melamine. Cyanuric

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acid concentrations are higher in bovine feed, which is reflected in the urinary concentration

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profiles in bovines.

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Country- and Gender-Related Variations in Concentrations. Creatinine concentrations in

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bovine urine collected from China, India and the United States ranged 4.5-360 (mean: 63), 4.9-

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170 (mean: 45) and 17-470 (mean: 89) mg/dL, respectively (Table S1). Significant positive

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correlations were found between the concentrations of melamine and creatinine (r2 = 0.311, p
> the United States (120 ng/g) > India (80 ng/g) (p < 0.01). A cyanuric acid

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concentration as high as 5,470 ng/g was found in a concentrated fodder sample, and a melamine

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concentration as high as 170 ng/g was found in a kaolin (feed additive) sample, from China.

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Because we had large number of samples collected in China, gender-specific differences in

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urinary concentrations of melamine and cyanuric acid were examined only for samples from this

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country. The median urinary concentration of cyanuric acid was significantly higher in cows

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(1,600 μg/g creatinine; Holstein) than in bulls (920 μg/g creatinine; Simmental) (Figure 2B; p
0.05) was observed in urinary melamine

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concentrations between genders (median: 15 vs 9.4 μg/g creatinine). The reason for this pattern is

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not known but likely due to dietary composition and/or metabolic differences between cows and

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bulls.

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nitrogen/cyanuric acid may be added in cow feed to increase the milk yield. No significant

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difference (p > 0.05) was observed in urinary melamine (median: 2.6 vs 3.1 μg/g creatinine) and

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cyanuric acid concentrations (140 vs 190 μg/g creatinine) between cows and buffalos from India.

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It is worth to mention that our analysis is tempered by the small sample size from each of the

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countries studied. Furthermore, our samples from China were from those commercial animal farms

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in which bovines were housed permanently in shelters and fed with the commercial feed. In other

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words, cows/bulls were not allowed to graze in pastures/grasslands. In contrast, samples from the

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United States and India were taken from bovines that were fed with a combination of

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grain/commercial feed and grass/hay, and were allowed to graze (i.e., free range) in pastures.

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Further studies with a large number of samples from different types of animal husbandry practices

It is possible that additives and concentrated fodder that contain high levels of


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are necessary to describe country-, gender-, age-, and breed-specific differences in the

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concentrations of melamine and cyanuric acid in bovine urine.

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Cumulative Daily Intake. Based on the measured urinary concentrations of melamine and

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cyanuric acid, we estimated their cumulative daily intakes (CDI; ng/kg bw/day) in bovines, using

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the following equation:

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CDI = (𝑈𝐶 ∗ 𝑈𝑉𝑒𝑥𝑐𝑟𝑒𝑡𝑖𝑜𝑛)/(𝑚 ∗ 𝑓),

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where UC is the urinary melamine or cyanuric acid concentration (ng/mL), UVexcretion is the daily

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urine excretion volume (20 L/day),43 m is the body weight (600 kg),44 and f is the fraction of

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melamine or cyanuric acid excreted in urine relative to the exposure dose. A value of 100% was

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used for f in this study, based on the results of previous toxicokinetic studies, which showed that

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rodents excrete > 90% of melamine unmetabolized in urine within 24 h of exposure.14, 34

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The calculated CDI values of melamine were in the ranges of 23–900 (median: 190), 7.9–270

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(28), and 16–300 (61) ng/kg bw/day for bovines from China, India and the United States,

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respectively (Figure 3A). Significantly higher CDIs of cyanuric acid were found at 240–1.8×105

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(median: 2.0×104), 120–1.1×105 (880) and 260–1.1×105 (5600) ng/kg bw/day for bovines from

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China, India and the United States, respectively (Figure 3B). The reported CDIs of ∑melamine

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were in the ranges of 2100–5710 ng/kw bw/day for dogs and 5830–9400 ng/kg bw/day for cats.36

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The reported CDIs of melamine and cyanuric acid in humans were in the ranges of 0.80–1130

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(mean: 66) and 32–4130 (mean: 320) ng/kg bw/day, respectively. Overall, the CDIs of melamine

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and cyanuric acid in bovines were 1−2 orders of magnitude higher than those reported for pet dogs

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and cats as well as humans.

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The World Health Organization (WHO) recommended a tolerable daily intake (TDI) of 2.0×105

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ng/kg bw/day for melamine and 1.5×106 ng/kg bw/day for cyanuric acid.45 These two TDI values



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have been suggested for humans. No TDI values are available for melamine and cyanuric acid

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exposure in bovines. The potential health risks from current melamine and cyanuric acid exposure

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in bovines were calculated as hazard quotients (HQs), which were the ratios of melamine or

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cyanuric acid exposure dose (i.e., CDI) divided by the corresponding TDI (Table 2). The mean

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HQs of melamine were 1.1×10-3, 4.3×10-4, and 2.1×10-4 for bovines from China, the United States,

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and India, respectively, and the corresponding values of cyanuric acid were 2.2×10-2, 1.1×10-2, and

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6.0×10-3, respectively. The calculated HQs for melamine and cyanuric acid were all well below

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1.0. These results suggest that the current exposure doses of melamine and cyanuric acid do not

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pose a risk to the health of bovines.

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Based on the CDI of melamine and cyanuric acid in bovines, we estimated the carryover of

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melamine and cyanuric acid from feed to dairy milk. For this calculation, daily milk yield from a

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cow was presumed at 28 L/day.43 For dairy cows exposed to melamine via feed, a transfer factor

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of 2.2% in milk was reported for this compound.33 No such experimentally determined transfer

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factor was available for cyanuric acid. Nevertheless, for the sake of simplicity, the transfer rates

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determined for melamine also was used for cyanuric acid. For this calculation, only data from the

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United States, which included all dairy cows, were used. On the basis of this exposure dose

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calculation, a daily dose of 5.1×104 ng of melamine and 8.7×106 ng of cyanuric acid (determined

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for cows from the United States) would correspond to concentrations in dairy milk of 0.04 and 6.8

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ng/mL, respectively. In our 2018 study, the concentrations of melamine and cyanuric acid at 1.5

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and 9.4 ng/mL, respectively, were found in dairy milk purchased in the United States.7 These

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results suggest that carryover of melamine from feed to milk was 2.7%, whereas that of cyanuric

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acid was 73%. Thus, melamine found in dairy milk purchased in stores originates from sources

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arising from production processes and packaging materials, whereas that of cyanuric acid is mainly


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from feed.46, 47 It should be noted, however, that pharmacokinetics of cyanuric acid in bovines have

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not been studied and that our calculations were not based on matched concentrations of urine and

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milk samples. This crude analysis, however, should shed light on the transfer factors of melamine

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and cyanuric acid from feed to milk.

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Our data provide the first evidence of the occurrence and transfer of melamine and cyanuric

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acid in bovines. We found significant country-specific differences in urinary concentrations of

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melamine and cyanuric acid in bovines. The estimated daily intakes of melamine and cyanuric

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acid in bovines were at least an order of magnitude below the current TDI. As indicated above,

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our study has some limitations, and, thus, the data should be interpreted within those confines.

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Further studies are needed to evaluate the toxicity of melamine and cyanuric acid in bovines.

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■ ASSOCIATED CONTENT

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Supporting Information

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Details of sample preparation and instrumental analytical method. Tables containing detailed

315

information on bovine urine (Table S1) and feed (Table S2) as well as corresponding target

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chemical concentrations. Multiple reaction monitoring (MRM) transitions of target chemicals

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(Table S3). Figure showing typical chromatograms of target chemicals in samples and analytical

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standards (Figure S1).

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■ AUTHOR INFORMATION

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Corresponding Author

322

*Tel.: +1 518 474 0015. Fax: +1 518 473 2895.

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E-mail: [email protected].



324

Notes

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The authors declare no competing financial interest.

326 327

Acknowledgements

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Authors are thankful to Mr. Jason Robertson, Ms. Cassandra Peterson and Mr. Adam Martin,

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Murray State University, for their help in collecting cow urine samples from Murray, KY.

330 331

REFERENCES

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(1) Sharma, K.; Paradakar, M., The melamine adulteration scandal. Food Secur. 2010, 2, 97-107.

333

(2) Ingelfinger, J. R., Melamine and the global implications of food contamination. New Engl. J.

334

Med. 2008, 359, 2745-2748.

335

(3) Gossner, C. M. E.; Schlundt, J.; Ben Embarek, P.; Hird, S.; Lo-Fo-Wong, D.; Beltran, J. J. O.;

336

Teoh, K. N.; Tritscher, A., The Melamine incident: Implications for international food and

337

feed safety. Environ. Health Perspect. 2009, 117, 1803-1808.

338

(4) Tittlemier, S. A.; Lau, B. P. Y.; Menard, C.; Corrigan, C.; Sparling, M.; Gaertner, D.; Pepper,

339

K.; Feeley, M., Melamine in infant formula sold in Canada: Occurrence and risk assessment.

340

J. Agric. Food Chem. 2009, 57, 5340-5344.

341 342

(5) Londono, V. A. G.; Punales, M.; Reynoso, M.; Resnik, S., Melamine contamination in milk powder in Uruguay. Food Addit. Contam. B 2018, 11, 15-19.

343

(6) EFSA. Scientific opinion on melamine in food and feed. EFSA J. 2010, 8, 1573-1717.

344

(7) Zhu, H. K.; Kannan, K., Continuing occurrence of melamine and its derivatives in infant formula

345

and dairy products from the United States: Implications for environmental sources. Environ.

346

Sci. Technol. Lett. 2018, 5, 641-648.

347

(8) Qin, Y. C.; Lv, X. W.; Li, J.; Qi, G. H.; Diao, Q. Y.; Liu, G. H.; Xue, M.; Wang, J. Q.; Tong, J.

348

M.; Zhang, L. Y.; Zhang, K. Y., Assessment of melamine contamination in crop, soil and

349

water in China and risks of melamine accumulation in animal tissues and products. Environ.

350

Int. 2010, 36, 446-452.


Page 16 of 25

Page 17 of 25


351

(9) Wu, C. F.; Peng, C. Y.; Liu, C. C.; Lin, W. Y.; Pan, C. H.; Cheng, C. M.; Hsieh, H. M.; Hsieh,

352

T. J.; Chen, B. H.; Wu, M. T., Ambient melamine exposure and urinary biomarkers of early

353

renal injury. J. Am. Soc. Nephrol. 2015, 26, 2821-2829.

354

(10) Zhu, H. K.; Kannan, K., Distribution profiles of melamine and its derivatives in indoor dust

355

from twelve countries and the implications for human exposure. Environ. Sci. Technol. 2018,

356

52, 12801-12808.

357

(11) Zhu, H. K.; Halden, R. U.; Kannan, K., A nationwide survey of the occurrence of melamine

358

and its derivatives in archived sewage sludge from the United States. Environ. Pollut. 2019,

359

245, 994-999.

360

(12) Zhu, H. K.; Lee, S.; Moon, H. B.; Kannan, K., Spatial and temporal trends of melamine and

361

its derivatives in sediment from Lake Shihwa, South Korea. J. Hazard. Mater. 2019, 373,

362

671-677.

363 364

(13) Richardson, S. D., Environmental mass spectrometry: Emerging contaminants and current issues. Anal. Chem. 2010, 82, 4742-4774.

365

(14) Panuwet, P.; Nguyen, J. V.; Wade, E. L.; D'Souza, P. E.; Ryan, P. B.; Barr, D. B.,

366

Quantification of melamine in human urine using cation-exchange based high performance

367

liquid chromatography tandem mass spectrometry. J. Chromatogr. B: Analyt. Technol.

368

Biomed. Life Sci. 2012, 887, 48-54.

369

(15) Sathyanarayana, S.; Flynn, J. T.; Messito, M. J.; Gross, R.; Whitlock, K. B.; Kannan, K.;

370

Karthikraj, R.; Morrison, D.; Huie, M.; Christakis, D.; Trasande, L., Melamine and cyanuric

371

acid exposure and kidney injury in US children. Environ. Res. 2018, 171, 18-23.

372 373

(16) Zhu, H. K.; Kannan, K., Inter-day and inter-individual variability in urinary concentrations of melamine and cyanuric acid. Environ. Int. 2018, 123, 375-381.

374

(17) Lam, C. W.; Lan, L.; Che, X. Y.; Tam, S.; Wong, S. S. Y.; Chen, Y.; Jin, J.; Tao, S. H.; Tang,

375

X. M.; Yuen, K. Y.; Tam, P. K. H., Diagnosis and spectrum of melamine-related renal disease:

376

Plausible mechanism of stone formation in humans. Clin. Chim. Acta 2009, 402, 150-155.

377 378

(18) Chu, C. Y.; Fung, K. P.; Wang, C. C., Effects of low-dose melamine exposure during pregnancy on maternal and fetal kidneys in rats. Environ. Toxicol. 2018, 33, 370-380.

379

(19) Li, G.; Jiao, S. F.; Yin, X. J.; Deng, Y.; Pang, X. H.; Wang, Y., The risk of melamine-induced

380

nephrolithiasis in young children starts at a lower intake level than recommended by the WHO.

381

Pediatr. Nephrol. 2010, 25, 135-141. 17 ACS Paragon Plus Environment


382

(20) Liu, C. C.; Wu, C. F.; Chen, B. H.; Huang, S. P.; Goggins, W.; Lee, H. H.; Chou, Y. H.; Wu,

383

W. J.; Huang, C. H.; Shiea, J.; Lee, C. H.; Wu, K. Y.; Wu, M. T., Low exposure to melamine

384

increases the risk of urolithiasis in adults. Kidney Int. 2011, 80, 746-752.

385

(21) Hsieh, T. J.; Hsieh, P. C.; Tsai, Y. H.; Wu, C. F.; Liu, C. C.; Lin, M. Y.; Wu, M. T., Melamine

386

induces human renal proximal tubular cell injury via transforming growth factor- and

387

oxidative stress. Toxicol. Sci. 2012, 130, 17-32.

388 389

(22) Bolden, A. L.; Rochester, J. R.; Kwiatkowski, C. F., Melamine, beyond the kidney: A ubiquitous endocrine disruptor and neurotoxicant? Toxicol. Lett. 2017, 280, 181-189.

390

(23) Yang, Y.; Xiong, G. J.; Yu, D. F.; Cao, J.; Wang, L. P.; Xu, L.; Mao, R. R., Acute low-dose

391

melamine affects hippocampal synaptic plasticity and behavior in rats. Toxicol. Lett. 2012,

392

214, 63-68.

393

(24) Chu, C. Y.; Tang, L. Y.; Li, L.; Shum, A. S. W.; Fung, K. P.; Wang, C. C., Adverse

394

reproductive effects of maternal low-dose melamine exposure during pregnancy in rats.

395

Environ. Toxicol. 2017, 32, 131-138.

396

(25) Xie, J. H.; Chen, D. D.; Wu, Q.; Wang, J.; Qiao, H., Spectroscopic analyses on interaction of

397

melamine, cyanuric acid and uric acid with DNA. Spectrochim. Acta A Mol. Biomol.

398

Spectrosc. 2015, 149, 714-721.

399

(26) Wu, C. F.; Hsiung, C. A.; Tsai, H. J.; Tsai, Y. C.; Hsieh, H. M.; Chen, B. H.; Wu, M. T.,

400

Interaction of melamine and di-(2-ethylhexyl) phthalate exposure on markers of early renal

401

damage in children: The 2011 Taiwan food scandal. Environ. Pollut. 2018, 235, 453-461.

402 403 404 405

(27) Wu, M. T.; Wu, C. F.; Chen, B. H., Behavioral intervention and decreased daily melamine exposure from melamine tableware. Environ. Sci. Technol. 2015, 49, 9964-9970. (28) Ibarra, V. G.; de Quiros, A. R. B.; Sendon, R., Study of melamine and formaldehyde migration from melamine tableware. Eur. Food Res. Technol. 2016, 242, 1187-1199.

406

(29) Chen, Y. Q.; Yang, W. J.; Wang, Z. Y.; Peng, Y.; Li, B.; Zhang, L. Y.; Gong, L. M., Deposition

407

of melamine in eggs from laying hens exposed to melamine contaminated feed. J. Agr. Food

408

Chem. 2010, 58, 3512-3516.

409

(30) Baynes, R. E.; Smith, G.; Mason, S. E.; Barrett, E.; Barlow, B. M.; Riviere, J. E.,

410

Pharmacokinetics of melamine in pigs following intravenous administration. Food Chem.

411

Toxicol. 2008, 46, 1196-1200.


Page 18 of 25

Page 19 of 25

412 413


(31) Lu, M. B.; Yan, L.; Guo, J. Y.; Li, Y.; Li, G. P.; Ravindran, V., Melamine residues in tissues of broilers fed diets containing graded levels of melamine. Poult. Sci. 2009, 88, 2167-2170.

414

(32) Buur, J. L.; Baynes, R. E.; Riviere, J. E., Estimating meat withdrawal times in pigs exposed to

415

melamine contaminated feed using a physiologically based pharmacokinetic model. Regul.

416

Toxicol. Pharmacol. 2008, 51, 324-331.

417 418

(33) Cruywagen, C. W.; Stander, M. A.; Adonis, M.; Calitz, T., Hot topic: Pathway confirmed for the transmission of melamine from feed to cow's milk. J. Dairy Sci. 2009, 92, 2046-2050.

419

(34) Mast, R. W.; Jeffcoat, A. R.; Sadler, B. M.; Kraska, R. C.; Friedman, M. A., Metabolism,

420

disposition and excretion of [14C] melamine in male Fischer 344. Food Chem. Toxicol. 1983,

421

21, 807-810.

422

(35) Zhang, M. Q.; Li, S. J.; Yu, C. Y.; Liu, G. Y.; Jia, J. Y.; Lu, C. A.; He, J.; Ma, Y. H.; Zhu, J.

423

M.; Yu, C., Determination of melamine and cyanuric acid in human urine by a liquid

424

chromatography tandem mass spectrometry. J. Chromatogr. B 2010, 878, 758-762.

425 426

(36) Karthikraj, R.; Bollapragada, R.; Kannan, K., Melamine and its derivatives in dog and cat urine: An exposure assessment study. Environ. Pollut. 2018, 238, 248-254.

427

(37) U.S. Food and Drug Administration. CFR-code of federal regulations. 2017.

428

https://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfCFR/CFRSearch.cfm?fr=573.220

429

(accessed Janurary 14, 2019).

430

(38) Xue, J. C.; Wu, Q.; Sakthivel, S.; Pavithran, P. V.; Vasukutty, J. R.; Kannan, K., Urinary levels

431

of endocrine-disrupting chemicals, including bisphenols, bisphenol A diglycidyl ethers,

432

benzophenones, parabens, and triclosan in obese and non-obese Indian children. Environ. Res.

433

2015, 137, 120-128.

434

(39) Dobson, R. L. M.; Motlagh, S.; Quijano, M.; Cambron, R. T.; Baker, T. R.; Pullen, A. M.;

435

Regg, B. T.; Bigalow-Kern, A. S.; Vennard, T.; Fix, A.; Reimschuessel, R.; Overmann, G.;

436

Shan, Y.; Daston, G. P., Identification and characterization of toxicity of contaminants in pet

437

food leading to an outbreak of renal toxicity in cats and dogs. Toxicol. Sci. 2008, 106, 251-

438

262.

439

(40) Zhu, X.; Wang, S.; Liu, Q.; Xu, Q.; Xu, S.; Chen, H., Determination of residues of cyromazine

440

and its metabolite, melamine, in animal-derived food by gas chromatography-mass

441

spectrometry with derivatization. J. Agric. Food Chem. 2009, 57, 11075-80.



442

(41) Sancho, J. V.; Ibanez, M.; Grimalt, S.; Pozo, O. J.; Hernandez, F., Residue determination of

443

cyromazine and its metabolite melamine in chard samples by ion-pair liquid chromatography

444

coupled to electrospray tandem mass spectrometry. Anal. Chim. Acta 2005, 530, 237-243.

445 446

(42) Ritota, M.; Manzi, P., Melamine detection in milk and dairy products: Traditional analytical methods and recent developments. Food Anal. Methods 2017, 11, 128-147.

447

(43) Spek, J. W.; Bannink, A.; Gort, G.; Hendriks, W. H.; Dijkstra, J., Effect of sodium chloride

448

intake on urine volume, urinary urea excretion, and milk urea concentration in lactating dairy

449

cattle. J. Dairy Sci. 2012, 95, 7288-7298.

450

(44) Buckley, F.; O'Sullivan, K.; Mee, J. F.; Evans, R. D.; Dillon, P., Relationships among milk

451

yield, body condition, cow weight, and reproduction in spring-calved Holstein-Friesians. J.

452

Dairy Sci. 2003, 86, 2308-2319.

453

(45) World Health Organization. Toxicological and Health Aspects of Melamine and Cyanuric

454

Acid. Report of a WHO Expert Meeting in Collaboration with FAO. Supported by Health

455

Canada. 2009. http://www.who.int/foodsafety/publications/chem/Melamine_report09.pdf

456

(accessed Janurary 14, 2019).

457 458

(46) Lund, K. H.; Petersen, J. H., Migration of formaldehyde and melamine monomers from kitchen- and tableware made of melamine plastic. Food Addit. Contam. 2006, 23, 948-955.

459

(47) Lu, J.; Xiao, J.; Yang, D. J.; Wang, Z. T.; Jiang, D. G.; Fang, C. R.; Yang, J., Study on

460

migration of melamine from food packaging materials on markets. Biomed. Environ. Sci.

461

2009, 22, 104-108.

462


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Table 1. Concentrations of melamine and its derivatives in bovine urine and feed collected from China, the United States and India. Bovine urinea (N1b=183; ng/mL) LOQsi dfj

MELd

AMDe

CYAf

0.20

0.10

0.12

100

45

100

∑3MELsg 100

Bovine feed (N2c=29; ng/g) MEL

AMNh

AMD

CYA

0.80

0.32

0.30

0.45

100

100

100

100

∑4MELs 100

min 0.70

Occurrence and Profiles of Melamine and Cyanuric Acid in Bovine

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