The study design for the CTEPP study has been discussed in-depth by Wilson et al. [17]. Briefly, the participants were recruited from six counties in NC from July 2000 to April 2001 and from six counties in OH from April 2001 to November 2001. The study cohort consisted of 256 preschool children; 129 children participated from NC and 127 children participated from OH. In NC, 66 children stayed-at-home with their adult caregivers during the day and 63 children attended daycare during the day. In OH, 69 children stayed-at-home with their adult caregivers during the day and 58 children attended daycare during the day. For the daycare group of children, environmental and personal samples were collected at both their homes and daycare centers. Environmental samples (soil, dust, outdoor air, and indoor air) and personal samples (hand wipes, solid food, and liquid food) were collected over a 48-h monitoring period at 129 homes and 13 daycare centers in NC and at 127 homes and 16 daycare centers in OH. Field staff collected environmental samples at both locations, and adult caregivers (i.e., parents and daycare teachers) collected personal samples from the children.

The CTEPP study was a human observational research study, as defined in 40 Code of Federal Regulations, Part 26.402 [22]. The study protocol and procedures used to obtain informed consent of the adult caregivers and the assent of the preschool children were approved by an independent institutional review board (Battelle) before beginning the study and complied with all applicable requirements of the Common Rule regarding additional protections for children (Subpart D). The study protocol and procedures were also approved by the US EPA’s Human Subjects Research Official prior to starting the study.

Detailed descriptions of the field sampling activities that occurred over a 48-h monitoring period at the children’s homes and/or daycare centers have been described in Wilson et al. [17]. The collection of the environmental samples and personal samples are briefly described below. Soil samples consisted of scraping the surface of a 0.1 m² area of bare soil with a putty knife (nearest a child’s play area) and placing it into a pre-cleaned glass jar. Indoor floor dust samples were collected from a 0.76 m² of carpeting from the child’s main activity area (e.g., living room or classroom) with a high volume surface sampler (Cascade Stack Sampling Systems, Bend, OR, USA) and transferring the sample to a pre-cleaned glass jar. Outdoor air was sampled over a 48-h period using a URG-2000 cartridge with a Thomas pump generating a flow rate of ~4.0 L/min. For indoor air, 48-h samples were collected using a URG-2000 cartridge with a SKC pump (~4.0 L/min) in the child’s main activity area. Hand wipe samples consisted of the adult caregivers wiping the front and back of both hands of the children with a 100 cm² pre-wetted cotton pad (SOF-WICK, Johnson and Johnson, Arlington, TX, USA) with 2 mL of 75% isopropanol and placing it into a pre-cleaned glass jar. Duplicate diet samples were collected from each child by their adult caregiver over the 48-h period; solid and liquid food samples were collected separately in 2 L pre-cleaned glass jars. Solid foods included all the fruits, vegetables, meats, dairy products, and desserts eaten by each child. Liquid foods included all of the beverages, excluding drinking water, consumed by each child. Examples of typical solid foods collected were apples, bananas, carrots, lunch meats, cheeses, and cookies, and examples of typical liquid foods collected were fruit juices, milk, and soft drinks. Duplicate amounts of solid and liquid foods were collected at homes, and duplicate serving of solid and liquid foods were collected at daycares. All samples were kept at reduced temperatures in coolers until picked up by field staff.

Detailed extraction and analytical procedures for the target pesticides in the environmental media and personal media can be found in Morgan et al. [18]. Briefly, the extraction methods for the OC, OP, and PY insecticides in each medium are as follows: soil samples (2 g) and dust samples (0.5 g) were sonicated with 10% diethyl ether in hexane, “concentrated” by Kuderna-Danish (KD) evaporation, followed by Florisil solid phase extraction (SPE), and concentrated again. Air samples and wipe samples were Soxhlet-extracted (~14-h) with dichloromethane (DCM), concentrated and subjected to Florisil SPE clean-up (if needed). Solid food samples were homogenized by a food chopper, and then 12 g of food were mixed with Extrelute and extracted using accelerated solvent extraction (ASE) with DCM, concentrated, and fractionated by gel permeation chromatography (GPC) with DCM, followed by an ENVI-Carb clean up. Liquid food samples (30 mL) were refluxed with DCM, filtered, concentrated, and then GPC clean-up with DCM. All sample extracts were adjusted to 1 mL with solvent and placed into glass vials. The extraction methods for the AC herbicides in each medium are as follows: soil samples (5 g) were mixed with Extrelute, extracted using ASE with acetone, and concentrated. Dust samples (0.5 g) were mixed with sand, then extracted using ASE with acetone, and concentrated. Air samples and hand wipe samples were Soxhlet-extracted with acetonitrile and concentrated. Solid food samples were homogenized with a food chopper and 8 g were mixed with extrelute and extracted using ASE with methanol, and concentrated. Liquid food samples (10 mL) were mixed with Extrelute and extracted using ASE with methanol, and concentrated. All extracts were reduced to 1 mL with solvent, derivatized with N-(tert-butyldimethylsilyl)-N-methyltrifluoroacetamine, and placed into glass vials.

The surrogate recovery standard used for the OC, OP, and PY insecticides was p,p’-DDE-d₄. The internal standards for the three classes of insecticides were phenanthracene-d₁₀/p,p’-dibromobiphenyl, diazinon-d₁₀, and p,p’-dibromobiphenyl, respectively. The surrogate recovery standard for the AC herbicides was 2,4-D-C₁₃, and the internal standard was dicamba-d₃. Matrix spikes were used for each target pesticide in all sampled media. All extracts were analyzed by a gas chromatograph with mass selective detection (6890/5973A Hewlett-Packard) in the selected ion monitoring (SIM) mode. Table 1 provides the estimated limits of detection (LODs) for the target pesticides in the environmental media and personal media. The estimated limit of detection (LOD) was defined “as the minimum analyte level detected in a sample (in a given medium) and was estimated to be one-half the limit of quantification (LOQ)” [23]. The estimated LOQ was about twice the LOD for each pesticide by matrix.

Field blanks for the pesticides were below the LODs in all media, except for chlorpyrifos and cis-permethrin in only 1 of 12 air samples each. The mean values of these two insecticides were below the LODs in the air samples, so no background corrections were made. Laboratory blanks for all pesticides were below the LODs in each sampled media. Relative percent differences between duplicate samples (aliquots of the same sample) for the target pesticides were less than 10% in all media, except for chlorpyrifos in the air samples (24%) and dust/soil samples (14%). Relative percent differences between the analytical duplicates (aliquots of the same sample extract) for the target pesticides were less than 8% in the sampled media. The mean recoveries for the surrogate recovery standards, p,p’-DDE-d₄ and 2,4-D-C₁₃, were between 73% and 100% and 75% and 91%, respectively, in all media. The matrix spikes for the OC, OP, and PY insecticides had mean recoveries in media from 71%–130%, except for diazinon (54%) and cyfluthrin (64%) in the liquid food samples and diazinon (58%) in the solid food samples. For the AC herbicides, the matrix spikes had mean recoveries in all media between 72% and 99%, except for the indoor and outdoor air samples (64%–69%).

Data values below the LOD were assigned the value of the LOD divided by the square root of two, except for the liquid food concentration data. Since the pesticide concentrations in the liquid food samples were barely detectable on the gas chromatographs, a more conservative value of LOD divided by the square root of 10 was used [18]. Descriptive statistics (frequency of detection, percentiles (50th and 95th), and range) were computed for the pesticides in each medium at both the homes and daycare centers.

The estimated potential exposures (ng/day) of the 129 NC children were calculated for “frequently detected” pesticides through the dietary, nondietary, and inhalation routes using equations reported in a previous article [18] and are presented in Table 2. A “frequently detected” pesticide was defined here as having at least a 50% detection frequency in two or more different sampled media. There were a total of eight pesticides that met this criteria: α-chlordane, γ-chlordane, heptachlor, chlorpyrifos, diazinon, cis-permethrin, trans-permethrin, and 2,4-D. In Table 2, the children’s potential intake doses (ng/kg/day) to the frequently detected pesticides were computed by dividing E_dietary, E_nondietary, or E_inhalation by their body weight (kg) and by a default absorption rate of 50%. We assumed a default 50% absorption rate for a pesticide by each route of exposure as little published data exist in humans [23,24]. The dermal route for the pesticides was not quantified for these children as past research has indicated that this is a minor exposure route [14,18,19]. In addition, the children’s estimated potential aggregate exposures and potential aggregate intake doses were calculated for frequently detected pesticides (chlorpyrifos, permethrin, and 2,4-D) that had measureable levels for all three exposure routes. All statistical analyses were performed using SAS Version 8.0 (SAS, Cary, NC, USA).

In this NC cohort of CTEPP preschool children, there were a total of 58 males and 71 females. The children’s median age was 47 months, and their ages ranged between 20 months and 66 months. The racial background of the children was reported as white (55%), black (37%), Hispanic (4%), other (3%), and unknown (1%). The majority of the children (61%) lived in homes with a total household income of less than $50,000 per year. The children’s median body weight was 16.7 kg, and ranged from 10.4 to 44.1 kg.

In the questionnaires, 74% and 38% of the 129 homeowners reported applying products containing insecticides and herbicides, respectively, since residing (≥1 year) at their residences. Of these homeowners, 90% had used products that contained insecticides and 88% had used products that contained herbicides within a year of field sampling at their homes. For the 13 daycares, 62% and 31% had applied products with insecticides and herbicides, respectively, in the past at their facilities (≥1 year). Of these daycares, 88% had used products with insecticides and 100% had used products with herbicides within a year of the field sampling.

Table 3 and Table 4 present the distributions of the 16 pesticides measured in the environmental media collected at 129 homes and 13 daycare centers in NC. The OC insecticides were detected in all sampled media, except for aldrin in soil samples and outdoor air samples (daycares, only). Among the measured OC insecticides, only α-chlordane and γ-chlordane were detected ≥50% in the dust, ndoor air, and outdoor air samples at both locations.

In addition, heptachlor was detected >50% in the outdoor air and indoor air samples at both the homes and daycare centers. The OP insecticides, chlorpyrifos and diazinon, were detected ≥50% in the dust, outdoor air, and indoor air samples at both settings. In particular at the homes, the median levels of chlorpyrifos were at least three times greater than the median levels of diazinon in the dust, outdoor air, and indoor air samples. For the PY insecticides, cis-permethrin and trans-permethrin were both detected ≥50% in the dust and indoor air samples at both locations. Cyfluthrin was detected >40% in only the dust samples in both settings. 2,4-D was the only AC herbicide that was detected >50% in the dust samples in both settings. Of the measured pesticides in the environmental media, the combined isomers of chlordane had the highest 95th percentile concentrations (≥25.0 ng/g) in soil samples at both the homes and daycare centers. For the dust samples, the median levels of the combined isomers of permethrin (>1,400 ng/g) were at least 10 times greater than the median levels for all of the other measured pesticides at both locations. Heptachlor had the highest median concentrations occurring among these pesticides in the indoor air samples (≥5.40 ng/m³) in both settings.

Table 5 and Table 6 provide the distributions of the 16 pesticides measured in the personal media at 129 homes and 13 daycare centers in NC. For the hand wipe samples, α-chlordane, γ-chlordane, chlorpyrifos, cis-permethrin, and trans-permethrin were detected above >50% in both settings. However, the median levels of cis/trans-permethrin were at least five times greater in the hand wipe samples than for all of the other measured pesticides. Chlorpyrifos and 2,4-D were detected above 50% in the solid food samples at the homes, and only chlorpyrifos was detected >50% in the solid food samples at the daycares. Lastly, none of the pesticides were detected often (<19%) in the liquid food samples in either setting.

The children’s estimated median potential exposures (ng/day) and potential intake doses (ng/kg/day) to the eight frequently detected pesticides through the dietary ingestion, nondietary ingestion, and inhalation routes are presented in Table 7. Also for comparison in Table 7, we have provided the established oral reference doses (RfD’s) and/or inhalation reference concentrations (RfC’s) for these pesticides that are available in the US EPA’s Integrated Risk Management System (IRIS) [25]. The estimated median potential intake doses of the children through the dietary ingestion route were the highest for the combined isomers of permethrin at 4.84 ng/kg/day and for 2,4-D also at 4.84 ng/kg/day. For the nondietary ingestion route, the children had the highest median potential intake dose of 2.39 ng/kg/day to the combined isomers of permethrin which was at least an order of magnitude higher than for the next highest pesticide, chlorpyrifos (0.156 ng/kg/day). In contrast, the children’s estimated median potential intake dose through the inhalation route was the most to heptachlor at 1.71 ng/kg/day, followed by chlorpyrifos at 1.42 ng/kg/day.

The children’s estimated potential aggregate intake doses by all three exposure routes were quantifiable for chlorpyrifos, cis/trans-permethrin, and 2,4-D and are depicted as a box-and-whiskers plot in Figure 1. The estimated median potential aggregate intake doses of the children were 4.6 ng/kg/day for chlorpyrifos, 12.5 ng/kg/day for cis/trans-permethrin, and 4.9 ng/kg/day for 2,4-D. At the 95th percentile, the children’s estimated potential aggregate intake doses were 31.7 ng/kg/day (chlorpyrifos), 397 ng/kg/day (cis/trans-permethrin), and 22.5 ng/kg/day (2,4-D). The results show that dietary ingestion (>60%) was the predominant route of the children’s exposures to all three pesticides.