Article pubs.acs.org/est
Cite This: Environ. Sci. Technol. XXXX, XXX, XXX−XXX
Parabens and Their Metabolites in Pet Food and Urine from New York State, United States Rajendiran Karthikraj,† Sonali Borkar,† Sunmi Lee,† and Kurunthachalam Kannan*,†,‡,§ †
Wadsworth Center, New York State Department of Health, Empire State Plaza, P.O. Box 509, Albany, New York 12201, United States ‡ Department of Environmental Health Sciences, School of Public Health, State University of New York at Albany, New York 12222, United States § Biochemistry Department, Faculty of Science and Experimental Biochemistry Unit, King Fahd Medical Research Center, King Abdulaziz University, Jeddah 21589, Saudi Arabia S Supporting Information *
ABSTRACT: The exposure of pets, such as dogs and cats, to a wide range of chemicals present in the indoor environment and the concomitant increase in noninfectious diseases in these companion animals are a concern. Nevertheless, little is known about the sources and pathways of exposure to chemicals in pets. In this study, we determined the concentrations of parabens in commercially available cat and dog foods as well as in urine samples from these pets collected from the Albany area of the state of New York in the United States. Parabens, especially methyl paraben (MeP), and their metabolites were found in all pet food and urine samples. The mean concentrations of total parabens (i.e., sum of parabens and their metabolites) in dog (n = 23) and cat (n = 35) food were 1350 and 1550 ng/g fresh wt, respectively. Dry food contained higher concentrations of parabens and their metabolites than did wet food, and cat food contained higher concentrations of target chemicals than did dog food. The mean concentrations of total parabens found in dog (n = 30) and cat (n = 30) urine were 7230 and 1040 ng/mL, respectively. In both pet food and urine, MeP (among parabens) and 4-hydroxy benzoic acid (4-HB) (among metabolites) were the dominant compounds. The metabolites of parabens accounted for ∼99% (∼99.1% in food and ∼98.9% in urine) of the total concentrations in both food and urine. The profiles of parabens and their metabolites in the urine of dogs and cats varied. In addition to diet, other sources of paraben exposures were found for dogs, whereas, for cats, the majority of exposures was identified as related to diet.
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INTRODUCTION Companion animals have been proposed as sentinels of human exposure to environmental chemicals because they share a living environment with humans and spend long hours indoors. Studies have shown that pets, particularly cats and dogs, are exposed to environmental contaminants present in the indoor environment.1−4 Exposure of cats and dogs in Japan, the United States, Sweden, the United Kingdom (UK), Pakistan, and Australia to polybrominated diphenyl ethers (PBDEs), with the highest levels in the blood of American cats and dogs, have been documented.5−12 Studies have linked chemical exposure in pets to several noninfectious diseases, such as diabetes, hypothyroidism, kidney diseases, and cancer, which have increased in cats and dogs in recent years.2,3,13−22 For example, over the past three decades, feline hypothyroidism has been one of the most common diseases in aged cats.14 Serum levels of organochlorine pesticides, polychlorinated biphenyls (PCBs), and PBDEs were found significantly higher in cats with pituitary tumors in comparison to healthy cats and cats with type 2 diabetes.9 Lea et al.23 showed an association between diethylhexyl © XXXX American Chemical Society
phthalate (DEHP) or polychlorinated biphenyl 153 (PCB 153) exposure and decline in sperm motility and increased cryptorchidism in dogs. The United States has the largest population of pets in the world.17 Based on a survey conducted by the American Pet Products Association (APPA), the average number of dogs and cats per U.S. household in 2016 was 1.49 and 2.00, respectively, with a total estimated population of 89.7 and 94.2 million, respectively.24 According to the APPA, in 2016, the annual cost spent on pets for food, supplies amd medicine, veterinary care, pet grooming, and boarding in the United States was $66.75 billion, of which 42.3% was spent on food.25 Pet foods are regulated by the U.S. Food and Drug Administration (FDA), and all pet food producers are required to display labels that indicate their pet food does not contain any harmful substances.26 Received: November 21, 2017 Revised: January 30, 2018 Accepted: February 12, 2018
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DOI: 10.1021/acs.est.7b05981 Environ. Sci. Technol. XXXX, XXX, XXX−XXX
Article
Environmental Science & Technology
cardboard box). One of the major ingredients listed for dry foods (75%) was chicken meat. The food samples were transferred into 50 mL polypropylene (PP) tubes and stored in a freezer at −20 °C until analysis. A total of 30 dog and 30 cat urine samples (N = 60) were collected from the Albany area of the state of New York from January through May of 2017. Dog and cat urine samples were provided by pet owners (17 dog and 2 cat), local veterinary hospitals (3 dog and 5 cat), and an animal shelter (10 dog and 23 cat). Based on the information provided by the owner, shelter, or hospital, the dogs and cats studied were fed with one or more of the pet foods analyzed in this study. All dog urine samples were collected directly using a polypropylene (PP) container without allowing for any possible contamination, and all cat urine samples were collected by cystocentesis or using a PP container. Information with regard to age, gender, and breed of the pets was obtained (Tables S1 and S2). Sample Extraction. A solid−liquid extraction (SLE) method was used for the extraction of parabens and their metabolites from pet foods. Briefly, pet food samples were homogenized (dry foods by a pestle and a mortar and wet foods by an ultraturrax disperser, IKA Works, Inc. Wilmington, NC), and ∼4 g for wet food and 2 g for dry food were transferred into a 15 mL PP tube. A known concentration of a mixture of labeled IS (20 ng each, except for 13C12-4-HB, which was spiked at 50 ng) was spiked, vortexed, and allowed to equilibrate for 1 h. To the spiked samples, 6 mL of ethyl acetate was added and shaken in a mechanical shaker for 45 min. The PP tubes were centrifuged at 4500g for 10 min, and the supernatant was transferred into another 15 mL PP tube. The extraction was repeated with 6 mL of ethyl acetate, and the supernatant was combined and concentrated to near dryness and then reconstituted with 1 mL of methanol. The extract was then kept at −20 °C for 1 h to separate the lipid layer. After centrifugation, the extract was filtered through a 0.2 μm nylon filter into a vial. Pet-urine samples were analyzed by following a method reported previously, with slight modifications.40 Briefly, 250 μL of urine was transferred into a 15 mL PP tube. Samples, procedural blanks, commercial urine (for matrix spike recovery studies), and the SRM (3672 and 3673) were spiked with a known amount of IS (20 and 50 ng) prior to extraction. The samples were then buffered with 500 μL of 1.0 M ammonium acetate that contained 22 units of β-glucuronidase and digested at 37 °C for 15 h in an incubator shaker. Thereafter, 1 mL of 0.2% acetic acid (pH ≈ 4) was added to each sample tube, and target analytes were extracted with 10 mL of ethyl acetate by shaking in a mechanical shaker for 2 h. The extracts were washed with 1 mL of Milli-Q water and centrifuged at 4500g for 10 min. The ethyl acetate layer was transferred into a 15 mL PP tube and concentrated to neardryness under a gentle nitrogen stream. Next, 250 μL of methanol was added, vortex-mixed, and transferred into a vial for HPLC−tandem mass spectrometry (MS/MS) analysis. Creatinine was measured in all pet urine samples by HPLC− MS/MS, as described elsewhere.41 The specific gravity of urine was measured using a refractometer (ATAGO, Tokyo, Japan). Creatinine concentrations and the specific gravity of pet urine samples are listed in Tables S1 and S2. Instrumental Analysis. A Shimadzu Prominence Modular HPLC system (Shimadzu Corporation, Kyoto, Japan), coupled with an API 3200 electrospray triple quadrupole mass spectrometer (ESI−MS/MS; AB Sciex, Framingham, MA) operated under the negative ion multiple reaction monitoring (MRM) mode, was used for the analysis of parabens and their
Nevertheless, pet food is one of the main sources of pet illness caused by microbial or chemical contamination.27 A few studies have reported the occurrence of contaminants in pet foods. Kang and Kondo28 determined the occurrence of bisphenol A (BPA) at concentrations of 13−136 ng/g in cat food and 11−206 ng/g in dog food. Abd-Elhakim et al.29 screened 20 commercial pet foods for heavy metals and found that the concentration of tin exceeded the safe level proposed by the European Commission (EC). In the present study, we determined the presence of parabens, which are commonly used as antimicrobial preservatives in pharmaceuticals and food, including pet food. Parabens are esters of p-hydroxy benzoic acid and are broadspectrum antimicrobial agents used in personal care products, pharmaceuticals, and food products.30−33 Both in vitro and in vivo studies have demonstrated that parabens are endocrinedisrupting chemicals (Darbre and Harvey, 2008).34 Exposure to parabens was linked to reproductive effects in men.35,36 The U.S. FDA and the European Food Safety Authority (EFSA) have set the permissible limits for parabens in human food products.37,38 Exposure of humans to parabens, however, is ubiquitous.39−41 Nevertheless, little is known on the exposure of pet animals to parabens and their derivatives.42 We determined the concentrations of parabens and their metabolites in cat and dog urine as well as pet food available commercially for these companion animals.
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MATERIALS AND METHODS Chemicals. Methyl paraben (MeP), ethyl paraben (EtP), propyl paraben (PrP), butyl paraben (BuP), heptyl paraben (HpP), and benzyl paraben (BzP) as well as five metabolites (namely, 4-hydroxy benzoic acid (4-HB), 3,4-dihydroxy benzoic acid (3,4-DHB), methyl protocatechuate (OH-MeP), ethyl protocatechuate (OH-EtP), and benzoic acid (BA)) were measured in this study. The analytical standards of MeP, EtP, PrP, BuP, HpP, BzP, and 4-HB were purchased from Accustandard, Inc. (New Haven, CT). OH-MeP, OH-EtP, 3,4-DHB, BA, creatinine (≥98%), formic acid (≥95%), and β-glucuronidase (from Helix pomatia; type HP-2, aqueous solution, ≥ 100 000 units/mL) were purchased from SigmaAldrich (St. Louis, MO). The isotopically labeled internal standards (IS), 13C6-MeP, 13C6-EtP, 13C6-PrP, 13C6-BuP, 13 C6-HepP, 13C6-BzP, and 13C6-4-HB, were purchased from Cambridge Isotope Laboratories (Andover, MA). D3-creatinine (IS) was purchased from CDN Isotopes (Pointe-Claire, Quebec, Canada). High-performance liquid chromatography (HPLC)grade solvents were purchased from Mallinckrodt Baker (Phillipsburg, NJ), and ultrapure water was obtained from a water purification system (Barnstead International; Dubuque, IA). Commercial urine (Surine Negative Urine Control) was purchased from Cerilliant (Round Rock, TX), and the urine standard reference material (SRM nos. 3672 and 3673) was purchased from the National Institute for Standards and Technology (NIST; Gaithersburg, MD). Sample Collection. A total of 23 dog and 35 cat foods were purchased (N = 58) from local stores in Albany, New York, during March and April of 2017. These foods represent 10 and 13 different brands of dog and cat food, respectively, and were made in the United States, Thailand, and Canada. The dog food included 7 dry foods (granules, n = 7) and 16 wet foods (granules, n = 3; broth, n = 13). Similarly, cat food included 13 dry foods (granules, n = 5; solid, n = 8) and 22 wet foods (granules, n = 8; broth, n = 14). All dry foods were packaged in plastic bags (except for a dog food that was packed in a B
DOI: 10.1021/acs.est.7b05981 Environ. Sci. Technol. XXXX, XXX, XXX−XXX
Article
Environmental Science & Technology metabolites in pet foods. The optimized chromatographic and mass spectrometric parameters have been described elsewhere.43 An API 4500 electrospray QTRAP mass spectrometer (ESI− MS/MS; Applied Biosystems, AB Sciex, Framingham, MA) interfaced with an Agilent 1260 HPLC (Agilent Technologies Inc., Santa Clara, CA) was used in the analysis of target chemicals in pet urine. For chromatographic separation of target chemicals, a Zorbax SB-Aq (150 mm × 2.1 mm, 3.5 μm; Agilent Technologies Inc., Santa Clara, CA) column serially connected to a Javelin guard column (Betasil C18, 20 × 2.1 mm, 5 μm) was used. Separation of target analytes was achieved using a mobile-phase gradient flow (Table S3). Quality Assurance and Quality Control. For every 10 samples, one sample was analyzed in duplicate. The concentrations measured in duplicate samples were within ±15% of the mean. A 12-point calibration curve with concentrations that ranged from 0.05 to 500 ng/mL and a regression coefficient (R) of >0.99 was used in the quantification of concentrations. The limits of quantification (LOQ) and matrix spike recoveries of target chemicals in pet foods are presented in Table S4. Several procedural blanks were analyzed to monitor for contamination that can arise from reagents and materials used in sample extraction. MeP (0.13 ng/mL), EtP (0.11 ng/mL), and BA (40.0 ng/mL) were present in procedural blanks, and these values were subtracted from the concentrations reported for samples. The measured concentration of parabens and their metabolites in pet foods are reported on a fresh weight basis. For every 15 urine samples, 2 procedural blanks, a matrix blank (commercial urine spiked with IS), and 2 matrix spikes (native standards spiked at two concentrations, 20 and 40 ng) were analyzed (Table S5). Methanol (as a solvent blank) and a midpoint calibration standard (QC check) were injected between every 10 samples to check for carryover (if any) between sample injections and drift in instrumental sensitivity. An isotope-dilution method was used for the quantification of parabens and their metabolites in pet urine. Samples were diluted and reinjected when the responses of the target analytes exceeded the calibration range of the instrument. The LOQs and recoveries (matrix spike and standard reference materials) of target analytes in pet urine are presented in Table S5. An empty PP container was extracted with methanol to test for any background contamination of target chemicals. No detectable concentrations of parabens or their metabolites were found in the container. Because most of the cat urine samples were collected by cystocentesis, the syringe and the needle used for sample collection were tested for background contamination, and no target chemicals were found. Data Analysis and Calculation. SPSS software (version 22.0) was used for statistical analysis. To compare the differences in concentrations between dogs and cats and among the three age groups of pets, a Student t test and one-way ANOVA tests were performed, respectively. The probability value of p ≤ 0.01 (99% confidence interval; two-tailed) was set for statistical significance. Microsoft Excel 2016 was used for the calculation of mean, median, SG-adjusted, and creatinineadjusted concentrations. We estimated the daily dietary intake (EDI) of parabens and their metabolites for dogs and cats on the basis of the concentrations measured in foods using eq 1:32
where EDI is the estimated daily intake through diet, C is the concentration of parabens and their metabolites found in pet foods (ng/g), DC is the daily food consumption rate (g/day; the average calculated food consumption rate based on the body weight, Table S6 and Table S7), and BW is the body weight (kg). Pet animals were classified and grouped based on their age, body weight, or both for the comparison of measured concentrations of target chemicals.22 Because the size (i.e., BW) of dogs can vary depending on the breed, based on the information available in the literature, we calculated an average BW for a given age and gender.44 Based on the BW, we grouped dogs into three categories (Table S6), i.e., small (5.9−7.7 kg), medium (17.2−25 kg), and large (34 kg and above). However, for cats, the BW of different breeds did not vary significantly with respect to age; hence, cats were grouped into three categories (Table S7) based on age and BW, i.e., 10−12 weeks (0.8−1 kg), 3−6 months (1.4−2 kg), and 1 year and above (2−6.8 kg). The average and worst-case scenarios of exposure were calculated based on the mean and 95th percentile concentrations, respectively, in foods. To assess the cumulative daily intake (CDI), urinary concentrations were extrapolated to estimate the total exposure dose of parabens.32 The following equation was used for the calculation of CDI: urinary concentration CDI =
The urine excretion volume for pets was calculated from their body weight (BW) (Table S8). The average urine excretion rates for dogs and cats were 20 and 28 mL/kg bw/day, respectively.45,46 Parabens do not significantly accumulate in tissues of mammals (rats, rabbits and humans) and readily hydrolyzed/metabolized and eliminated in urine with
