Journal of Exposure Science and Environmental Epidemiology (2007) 17, 559–566 r 2007 Nature Publishing Group All rights reserved 1559-0631/07/$30.00 www.nature.com/jes Identification of human urinary metabolites of acetochlor in exposed herbicide applicators by high-performance liquid chromatography-tandem mass spectrometry DANA B. BARRa, CYNTHIA J. HINESb, ANDERS O. OLSSONa, JAMES A. DEDDENSb,c, ROBERTO BRAVOa, CYNTHIA A.F. STRILEYb, JESSICA NORRGRANa AND LARRY L. NEEDHAMa aCenters for Disease Control and Prevention, National Center for Environmental Health, Atlanta, Georgia, USA bCenters for Disease Control and Prevention, National Institute for Occupational Safetyand Health, Cincinnati, Ohio, USA cDepartment of Mathematical Sciences, Universityof Cincinnati, Cincinnati, Ohio, USA Acetochlor is a preemergent chloroacetanilide herbicide used to control annual grasses and small-seeded broadleaf weeds. It is the second most abundantly applied herbicide on corn crops in the United States; however, human metabolites associated with known exposure to acetochlor have not been positively identified and confirmed. We positively identified acetochlor mercapturate (ACM) as a metabolite of acetochlor in urine samples collected during a 24-h period from custom (commercial) applicators who had applied acetochlor on either the day of or the day before urine collection. Concentrationsin applicator urine samplesrangedfrom 0.5 to 449 mg/l (0.3–121 mg/g creatinine). We found that ACM accounted for asmuch as42% of the total acetochlor-derived metabolites; however, as the exposure level decreased (based on total acetochlor metabolite level), ACM became a less abundant metabolite of acetochlor (o17%). Unmetabolized acetochlor was also measured in the urine samples analyzed. At high exposures (classified as 4100 mg/l), acetochlor accounted for about 0.8% of the total excreted acetochlor metabolites( B2% of the ACM concentrations). At lower exposures (classified as ACM o10 mg/l), ACM and acetochlor concentrationswere similar. Additionally, we tentatively identified another acetochlor metabolite that appeared to be important at low levelsof exposure. Journal of Exposure Science and Environmental Epidemiology (2007) 17, 559–566; doi:10.1038/sj.jes.7500583; published online 30 May 2007 Keywords: acetochlor, acetochlor mercapturate, biological monitoring, urine, mass spectrometry. Introduction indicated that the amount of alachlor used in 1993 (32.1 million pounds) was comparable to the amount of acetochlor Acetochlor, (2-chloro-N-(ethoxymethyl)-N-(2-ethyl-6-methyl- used in 2003. Mean application rates for acetochlor and phenyl)-acetamide), isa preemergent chloroacetanilide alachlor from 1994 to 2003 were similar (range 1.69–2.00 lbs/ herbicide used to control annual grasses and small-seeded acre and 1.61–2.16 lbs/acre, respectively) (USDA, 1994, 1995, broadleaf weeds(Monaco et al., 2002; Weed Science Society 1996, 1997, 1998, 1999, 2000, 2001, 2002, 2003, 2004). of America: Herbicide Handbook 2002). Acetochlor was Acetochlor inducesnasaltumorsin ratsat the maximum- registered for use on corn in the United States in 1994 (US tolerated dose of 1000 parts per million and is classified as EPA, 1994), largely replacing the use of the herbicide ‘‘likely to be carcinogenic to humans’’ by the US EPA alachlor on corn crops. After atrazine, acetochlor is the (Ashby et al., 1996; Dearfield et al., 1999). In human and rat second most abundantly applied herbicide on corn in the liver microsomes, acetochlor is bioactivated via a multistep United States(USDA, 2004). A 2003 survey of 92% of the pathway to the putative DNA-reactive metabolite, 2-methyl- US corn acreage indicated that 36.1 million poundsof 6-ethylbenzoquinoneimine (Jefferies et al., 1998; Coleman acetochlor had been applied to corn, whereasonly 2.6 million et al., 2000; Green et al., 2000). Exposure to acetochlor has pounds of alachlor were used (USDA, 2004). By contrast, a been reported to significantly increase estrogen-binding 1993 survey of 90% of the US corn acreage (USDA, 1994) activity in rat uteri (Rollerova et al., 2000). Acetochlor is structurally similar to the herbicides alachlor and metolachlor (Figure 1). Glutathione-derived mercaptu- 1. Address all correspondence to: Dr. Dana B. Barr, Centers for Disease rate metabolitesof alachlor and metolachlor have been Control and Prevention, 4779 Buford Highway, Mailstop F17, Atlanta, identified asimportant human urinary metabolitesafter GA 30341, USA. Tel.: 770-488-7886. Fax: 770-488-0142. E-mail: [email protected] occupational exposures (Driskell et al., 1996; Driskell and Received 6 October 2006; accepted 17 April 2007; published online 30 Hill Jr., 1997). By analogy, acetochlor mercapturate (ACM) May 2007 hasbeen hypothesizedto be a human urinary metabolite of Barr et al. Urinary acetochlor metabolitesin exposedapplicators O O O Cl O Cl Cl O N N O N NH2 Acetochlor Metolachlor Alachlor N-(ethoxymethyl)-2- C H ClNO C H ClNO C H ClNO ethyl-6-methylaniline 14 20 2 15 22 2 14 20 2 C H N 269.77 283.80 269.77 9 13 135.21 O OH O S O N NH O NH O Metabolite A Acetochlor Mercapturate N-(ethoxymethyl)-2- C19H28N2O5S ethyl-6-methylaniline 396.50 C12H19NO 193.29 Figure 1. Structuresof acetochlor, related herbicidesand acetochlor metabolitesor degradates. acetochlor (Swan et al., 2003; Olsson et al., 2004; Curwin (CDC) collected urine, air, dermal patch, hand rinse and et al., 2005). Human exposure to alachlor has also been saliva samples from 15 male custom (or commercial) estimated by quantifying the hydrolysis product of alachlor- applicatorsin Ohio who applied herbicidesin the spring to related metabolites, 2,6-diethylaniline, in urine (Driskell corn and soybean fields (Hines et al., 2001, 2003). Samples et al., 1996). The major portion of acetochlor metabolites were collected from each applicator at approximately 4-day in the urine may be similarly estimated by quantifying the intervals over six consecutive weeks for a total of 89 hydrolysis product, 2-ethyl-6-methylaniline (EMA). EMA applicator days, with 5–7 sampled days per applicator. Each will not represent an estimate of the total acetochlor custom applicator sprayed, on average, 1372.8 (range 9–17) metabolite concentration because hydroxylation of the ethyl different herbicides, and as a group, sprayed more than 30 chain on the phenyl ring hasbeen reported (Chloroacetani- different herbicides(Hines et al., 2003). Applicatorsworked lides, 1998). These metabolites will not be accounted for by full-time (mean 12.372.9 h per day) and up to 7 daysper EMA, but in this paper we will still consider EMA an week. Participation wasvoluntary and informed consentwas estimate of the total metabolite concentration. obtained. Thisstudycomplied with all national and The aim of our study was to identify metabolites resulting international guidelinesfor the protection of human research from occupational exposures to acetochlor and to quantify subjectsandwasapproved by the NIOSH Human Subjects these metabolites in 24-h applicator urine collections. We Review Board. positively identified one acetochlor metabolite in most of the urine samples analyzed and tentatively identified an addi- Sample Collection tional metabolite. In addition, we estimated total acetochlor On each sampling day, applicators collected all urine starting metabolite concentrations by measuring EMA after base with that day’sfirst-morning void (day 0) through to the hydrolysis. first-morning void of the next day (day 1). The volume of each sample was recorded. Occasionally, applicators either did not have any urine or forgot to collect their urine during Materials and methods one of the time periods. Because of the repeated measures design of the study, multiple urine samples were collected on StudyPopulation the same person, both within a day and across days. Samples In 1996, as part of a herbicide exposure assessment study, the were frozen in the field on dry ice and stored at À701Cuntil National Institute for Occupational Safety and Health analysis.Detailsof the samplingand field procedureshave (NIOSH) at the Centers for Disease Control and Prevention been reported previously (Hines et al., 2003). Acetochlor was 560 Journal of Exposure Science and Environmental Epidemiology (2007) 17(6) Urinary acetochlor metabolitesin exposedapplicators Barr et al. ab H H HO N HO N O O O O O Cl In source O N S fragmentation S O O N N Acetochlor C H ClNO O 14 20 2 C H N O S [M]+ =270 m/z 17 23 2 4 [M]+ =351m/z Acetochlor Mercapturate C19H28N2O5S 395 amu O H Cl N H H N HO N N O O C12H15ClNO C9H10N C5H8NO3 C10H14N + + [M]+ =224m/z [M]+ =133 m/z [M+H] =130 m/z [M] =148 m/z Figure 2. MS/MS analysisscheme for acetochlor (a) and itsmercapturate metabolite (b). Q ¼ quantification ion; C ¼ confirmation ion; amu ¼ atomic mass unit commonly substituted for alachlor during the study period. by a second analysis including confirmatory ions (Figure 2). Metabolitesof alachlor and metolachlor were found in many The method limit of detection (LOD) in the repeat analyses of the applicator urine samples (Hines et al., 2003); thus, we was0.09 mg/l. also expected to find acetochlor metabolites. The number of A set of four urine samples representing higher level poundsof acetochlor sprayed on day 0 and on the day before exposures, three urine samples representing lower level day0(dayÀ1) wasalsorecorded for each applicator day. exposure, and four ‘‘blank’’ urine samples were analyzed to estimate total metabolite concentration and for metabolite LaboratoryAnalysis identification. The classification of exposure level was All urine samples collected on days where the applicator determined based on their urinary ACM concentrations sprayed acetochlor on either day 0, day À1orbothdayswere (high 4100 ng/ml; low o1.5 ng/ml; and ‘‘blank’’ o0.09 mg/l). analyzed for acetochlor metabolites( n ¼ 59 of 408 samples Furthermore, these samples had little or no metolachlor collected) at the National Center for Environmental Health mercapturate that could potentially lead to overestimation of (NCEH) at the CDC. Six duplicate (samples split directly total acetochlor metabolitesbecauseEMA can be derived after application) samples were included among the samples from metolachlor metabolitesaswell asacetochlor metabo- shipped.