Urinary biomonitoring data for 2,4-D are available from several studies of both general population adults and children and from studies of farmers and farm family members:
The National Center for Environmental Health of the Centers for Disease Control and Prevention (CDC) measured 2,4-D in urine samples collected from a complex, stratified random sample of the civilian, non-institutionalized population of the U.S., ages 6 to 59, during 2001-2002, as part of the National Health and Nutrition Examination Survey (NHANES) (CDC 2005).
Morgan et al. (2004, 2008) recently examined the exposures of 135 preschool children and their adult caregivers to 2,4-D at their homes in North Carolina and Ohio from the Children’s Total Exposure to Persistent Pesticides and Other Persistent Organic Pollutants (CTEPP) study. Participants were randomly recruited from homes in six North Carolina and six Ohio counties. Participants were recruited by field staff from homes between February 2000 and February 2001 in NC and January 2001 and November 2001 in Ohio. Monitoring was performed over a 48-hour period at the participants’ homes. Spot urine samples and environmental samples including air, soil, dust, hand wipes and food were collected and analyzed for 2,4-D.
Alexander et al. (2007) reported urinary 2,4-D data from the Farm Family Exposure Study. Participants in the study included 34 farmers in Minnesota and South Carolina who were licensed applicators and their spouses and children (n=53) living on the farm property. Participants collected 24-hour urine samples the day prior to, the day of, and for 3 days following application of 2,4-D on their farms during the 2000 or 2001 growing season.
Curwin et al. (2005) measured urinary 2,4-D concentrations in 16 farmers 1 to 5 days following their application of 2,4-D on the farm during the spring and summer of 2001. Samples were composited from urine samples collected in the evening and the following first morning sample.
The Pesticide Exposure Assessment Study measured the extent to which agricultural pesticide applicators and their families in Ontario, Canada are exposed to pesticides during normal handling practices (Arbuckle et al. 2002, 2004; Arbuckle and Ritter 2005). Farmers from the previously conducted Ontario Farm Family Health Study (Arbuckle et al. 1999), who had reported using phenoxyacetic acid herbicides were telephoned in early 1996 to determine their eligibility for the current study. To be eligible, the farmer had to: 1) be planning to use 2,4-D or (4-chloro-2- methylphenoxy) acetic acid (MCPA) in the coming growing season, 2) be the individual who would be handling the herbicides on the farm, 3) have his or her home on the farm property, and 4) be currently living with his or her spouse. A total of 126 families provided a spot urine sample prior to handling either 2,4-D or MCPA and then provided two consecutive 24-hour samples following use of the herbicide. All samples were collected in 1996.
The Agricultural Health Study/Pesticide Exposure Study (AHS/PES) was designed to evaluate exposure to 2,4-D and chlorpyrifos in a subset of individuals enrolled in the Agricultural Health Study, which is a large, prospective epidemiological study of pesticide applicators and their spouses in Iowa and North Carolina to study the Page 8 of 31 relationships between agricultural exposures and disease. Participants in the AHS were contacted randomly and surveyed to ascertain their planned use of the 2,4-D and chlorpyrifos, and then a subset of participants were enrolled in the Pesticide Exposure Study (Thomas et al. 2009). Urinary samples were collected during 2001 and 2002, and included a preapplication first morning void sample, as well as a 24-hour sample starting the day of application (“Day 1”) and, optionally, for days 2 through 5 as well.
Descriptions of the Institutional Review Board (IRB) approvals and informed consent information for each of these studies are presented in the underlying publications.
Reference Doses and Biomonitoring Equivalents
The U.S. EPA recently conducted a review of 2,4-D and adopted both a chronic oral RfD as well as acute RfDs (applicable to single day exposures) for this herbicide (U.S. EPA 2004). The derivations of the BE values associated with the RfD values are summarized in Table 1. BEs are defined as the concentration of a chemical or its metabolite in a human biological medium (usually blood or urine) that is consistent with existing exposure guidance values. BE values are screening values corresponding to existing risk assessments and not intended for use as definitive measures of risk for individuals. A full description of the BE approach and application is beyond the scope of this review, but is presented elsewhere (see Hays and Aylward, 2009; Hays et al. 2007, 2008).
The pharmacokinetics of 2,4-D have been studied in two sets of human volunteers (Kohli et al. 1974; Sauerhoff et al. 1977). Both found that 2,4-D is eliminated in urine either as Page 9 of 31 the unchanged parent compound (80-95%) or as a conjugate, with urinary half-lives on the order of 1 day. There was no evidence of oxidative metabolism, consistent with data from other mammalian species (Timchalk 2004). Based on these pharmacokinetic data, continuing exposure for more than 1 week of exposure would result in a steady-state in which the amount excreted daily in urine would be approximately equivalent to the amount absorbed each day.
Because 2,4-D is excreted as the parent compound in urine, most biomonitoring evaluations of exposure to 2,4-D have relied on measurements (quantifying both free and conjugated parent compound) in urine samples (Knopp and Glass 1991; Knopp 1994; CDC 2005), although a few kinetic studies have examined plasma concentrations of 2,4- D in humans and animals as well (Kohli et al. 1974; Saghir et al. 2006; Sauerhoff et al. 1977; van Ravenzwaay et al. 2003). The relative ease of collection of urine samples compared to blood samples contributes to this choice. From a toxicological point of view, plasma concentrations of 2,4-D are probably more informative for predicting target tissue concentrations and responses (for example, neurotoxic responses). This would be particularly true under conditions of episodic, higher-level exposures. However, under conditions of chronic, low-level exposures, urinary excretion rates of 2,4-D should be specific and quantitatively relevant in a framework of a mass-balance assessment. That is, under exposure conditions that approximate steady-state conditions (consistent with the definition of chronic RfDs and related exposure guidance values; see, for example, the definition of reference dose provided under the U.S. EPA Integrated Risk Information Page 10 of 31 System (IRIS) program (U.S. EPA 2009) daily urinary excretion of 2,4-D should equal daily intake.
The straightforward elimination kinetics of 2,4-D (as parent compound or conjugate in urine with essentially no oxidative metabolism) and the lack of direct relationship between urinary concentration and critical internal dose metrics suggests a simple massbalance approach for derivation of BE values for urinary 2,4-D consistent with chronic exposure at the chronic RfD. The process of deriving the BEPOD and BERfD values for 2,4-D is detailed in Aylward and Hays (2008) and summarized below and in Table 1.
The point of departure (POD) for the U.S. EPA chronic RfD is a no-observed-adverseeffect- level (NOAEL) of 5 mg/kg-d in rats fed 2,4-D chronically in the diet. Applying an uncertainty factor (UF) of 10 for interspecies variation, the human equivalent POD is 0.5 mg/kg-d. Calculating the average concentration of 2,4-D in urine in humans associated with this chronic daily dose (after application of the interspecies UF) yields the BEPOD. The daily mass intake at the human equivalent POD was estimated for a variety of child and adult body weights. Estimated distributions of daily creatinine excretion or urinary volume as a function of sex, age, and body size were used in a Monte Carlo analysis to estimate a distribution of creatinine-adjusted urinary 2,4-D concentrations for various age and sex categories (methods are described in detail in Aylward and Hays, 2008). The average of median estimated creatinine-adjusted 2,4-D concentration consistent with chronic exposure at the human equivalent POD (the BEPOD) for 2,4-D for adults (males and females) is approximately 20,000 μg/L or 30,000 μg/g creatinine. These values were Page 11 of 31 consistent with the range of median values identified in the simulations for children of various ages. Concentrations at the 95th percentiles of the estimated distributions were generally within a factor of 2 of the median values.
The BE associated with the chronic RfD was derived by dividing the BEPOD by the UF of 10 for intraspecies variation and the UF of 10 applied by U.S. EPA for database uncertainties (for a total composite UF of 1,000 applied to the animal NOAEL POD). BE values corresponding to the acute RfDs were derived in a similar fashion, except that the assumption of steady-state was not made. Based on the urinary elimination half-life of approximately 1 day, an assumption was made that one-half of the intake doses at the human equivalent POD for the acute RfD values would be eliminated in the first 24 hours following exposures. Average urinary 2,4-D concentrations (both absolute and creatinine-adjusted) corresponding to one-half the human equivalent POD doses were estimated, and the intra-species and database uncertainty factors were then applied to obtain the BERfD_acute values. These BE values are appropriate for use when the exposure is short term and episodic and the timing of the sample collection compared to exposure is known. The derivation and resulting values are summarized in Table 1.