Data were collected as part of a larger study of pesticide exposure. Data collection was completed in 11 counties with large migrant and seasonal farmworker populations: Bladen, Columbus, Edgecombe, Greene, Harnett, Johnston, Lenoir, Pitt, Sampson, Wayne and Wilson Counties. Conservative estimates for 2007 by the North Carolina Employment Security Commission put the number of migrant farmworkers in these counties without H2A visas at 13,675 (36.2% of the 37,610 in North Carolina), the number of migrant farmworkers with H2A visas at 2,995 (34.3% of 8,730), and the number of seasonal farmworkers at 5,800 (22.8% of 25,407). The major hand-cultivated and hand-harvested crops in these counties are tobacco, sweet potatoes, and cucumbers.
A two-stage procedure was used to select farmworkers. First, three local agencies that provide health and social services to farmworkers prepared a list of farmworker residential sites or camps in the counties they served. Camps were approached in order until each agency recruited an assigned number of camps and a specified number of participants. All camps that were approached agreed to participate. Second, participants in each of the camps were recruited to participate. In camps with seven or fewer residents, all farmworkers were invited to participate. In camps with more than seven residents, eight to ten farmworkers were recruited with interviewers recruiting participants as they became available. The final sample consisted of 288 workers recruited at 41 camps. For this analyses workers not born in Mexico (n=15) and for whom metals data were not collected (n=15) were not included, producing a final sample size of 258.
Data collection relevant to this analysis included a detailed interview and a first morning urine void to measure metals. Participants were given an incentive valued at $20. Data used in this analysis were collected from May through June 2007. Data collectors included eight fluent Spanish speakers. All of the interviewers completed an intensive course of training that included a thorough review of camp and participant selection, recruitment procedures, and data collection procedures. Particular attention was directed toward the protection of human subjects, obtaining informed consent, and maintaining confidentiality. Interviewers were trained in safe procedures for the collection and handling of biological samples. Each team of interviewers was accompanied by a supervisor to help ensure that data collection procedures were properly followed.
A detailed interview was completed with the farmworker participants. The questionnaire included items on participant personal characteristics (e.g., age, educational attainment, place of origin), work experience in US and home country agriculture, current health status, and smoking history. Based on previous data collection in this population (Spangler et al., 2003
), which showed very low frequency of tobacco use in any other form, questions were restricted to cigarettes. The questionnaire was developed in English and translated by an experienced translator who was a native Spanish speaker familiar with Mexican Spanish. The translated questionnaire was reviewed by four fluent Spanish speakers familiar with farm work. The questionnaire was then pre-tested with 16 Spanish-speaking farmworkers and revised as needed.
At the end of each interview the interviewer gave the participants urine collection containers with labels attached. The urine collection and storage containers were provided by the Centers for Disease Control and Prevention and were certified to be free of metals. Participants were instructed to fill the containers with their first void upon rising the next morning. They were assured that the urine samples would be tested for agricultural chemicals and metals only, and not for the use of alcohol, drugs, or any health conditions. They were asked specifically to only provide their urine in the containers, not that of any other workers in the camp. They were asked specifically not to put any other fluid in the urine containers (e.g., water). Finally, they were asked specifically not to put any other substances in the urine containers. The participants from each camp placed their urine containers in a cooler with blue ice that was provided to them. Each morning a project interviewer stopped by the camp interviewed the previous evening and retrieved the containers, transported them to the nearest of the three collaborating community partners, transferred the samples into labeled containers, and placed the samples in a laboratory freezer where they were stored at −20°C.
All laboratory analyses were carried out by RTI International in Research Triangle Park, North Carolina. Sample digestion was carried out using a DigiPreP MS (SCP Science, Champlain, NY). Both an Analog Vortex Mixer (VWR, Suwanee, GA) and a Vortex Genie (Fischer Scientific, Pittsburgh, PA) were used for mixing the urine samples. A quadrupole ICP-MS X Series II X0637 (Thermo, Waltham, MA) was employed for the determination of the trace metals in urine. This instrument was equipped with a collision cell to mitigate the impact of polyatomic interferences. A low-volume impact bead spray chamber and Xt cones from the instrument manufacturer were used for all analyses.
Ultrex HNO3 and Ultrex 30% H2O2 (JT Baker, Phillipsburg, NJ) were used in the creation of standards and samples. All metals for internal standards and for calibration standards were National Institute of Standards and Technology (NIST)-traceable (High Purity Standards, Charleston, SC). A standard reference material (SRM 2760) (Toxic Metals in Freeze-Dried Urine) was obtained from NIST (Gaithersburg, MD). Distilled de-ionized (DDI) H2O (18 MΩ, Pure Water Solutions) was used for all sample and standard preparations.
Standard solutions for the calibration curve included all the analytes and their internal standards. Bismuth at 209 m/z was used as the internal standard for Pb and Hg. Cd’s internal standard was 89Y. For As, which was analyzed with the collision cell, 141Pr was the internal standard. All internal standards were prepared in one solution, at 1 ppm each, with 2% HNO3. The ICP-MS rinse solution contained 5% (v/v) HNO3 and 10 µg/mL Au.
The urine samples were thawed, and then vortexed for 10–15 seconds to ensure homogeneity. Two mL of each sample were aliquoted into a digestion tube, and HNO3 (1.00 mL), 1000 µg/mL Au (0.050 mL), and H2O2 (0.250 mL) were added. The samples were then digested by the DigiPreP MS heating block using the following sequence: ramp from room temperature to 60°C for 30 minutes, hold at 60°C for 30 minutes, ramp to 80°C for 10 minutes, hold at 80°C for 50 minutes, ramp to 110°C for 20 minutes, hold at 110°C for 100 minutes.
The digestion step decreased salt and organic interferences in the urine matrix. While simple dilutions (Townsend et al., 1998
; Goullé et al., 2005
; De Boer et al., 1998
) have been successfully employed for ICP-MS preparation, the acid digestion helped improve sample throughput by reducing deposits on the ICP-MS cones. After digestion was complete, the samples were brought up to a volume of 10.0 mL with DDI H2
O and then vortexed again. IS solution (0.050 mL) was added to 5.00 mL of the digested samples, and solutions were vortexed before ICP-MS analysis.
Limits of detection (LOD) were: 0.029 µg/L for As, 0.0001 µg/L for Cd, 0.014 µg/L for Hg, and 0.0001 µg/L for Pb.
Age was categorized as 18–24, 25–29, 30–39, and 40 or more years. Current smoker was dichotomized as yes or no, based on a question asking if a worker had smoked any cigarettes in the past month. Smoking was converted to pack years. Pack years were derived from the average number of packs per day reported, multiplied by the number of years smoked. For current smokers, additional data were available concerning the number of days per week the worker currently smoked. These data were incorporated into the pack year estimate for current smokers. Pack years were divided into ordered categories: <1 pack year, 1 to 5 pack years, and >5 pack years. Region of birth in Mexico was categorized using state of birth as Northern (Baja California, Baja California Sur, Chihuahua, Coahuila, Nuevo Leon, Sonora, Tamaulipas, and Durango), Southern (Campeche, Chiapas, Quintana Roo, Tabasco, and Yucatan), and Central (remaining states and the Federal District). Years worked in US agriculture was categorized as 1 or less, 2 to 3, 4 to 7, and 8 or more years.
Descriptive statistics were examined for all farmworkers in the sample that originated from Mexico and for whom metals data were available. Farmworker characteristics of interest (age, sex, region from Mexico, current smoker, pack-years, agriculture in home country, and years worked in US agriculture) were categorized into groups and frequency distributions reported.
Seventeen values for Hg were less than the LOD. These were imputed as LOD/sqrt(2) (Homung and Reed, 1990
). To adjust for the potential effect of dehydration, all the outcome measures are defined as microgram of metals per gram of creatinine. All metals data had skewed distributions; therefore log-transformations were used to normalize the data in order to conduct statistical analyses. Furthermore, all data analyses accounted for the study’s clustered design. Urinary metals data from the 2003–2004 National Health and Nutrition Examination Survey (NHANES) were used as reference data. As such, these NHANES data were used only to categorize the study data from farmworkers; the NHANES were not in any way meant to be baseline or optimal values. Rather, they were intended to indicate the levels of metals typical of US residents. The NHANES reference data were restricted to persons 18 years and older. Geometric means and confidence intervals for the comparison of NHANES data and the North Carolina farmworker sample were obtained using SAS callable SUDAAN (RTI International, Research Triangle Park, NC). Statistical tests were also conducted for each metal of interest to describe the associations between the metal concentrations and farmworker characteristics. A multivariate regression model was fitted for each continuous, log-transformed metal using PROC MIXED. The models incorporated farmworker characteristics of interest including age, years worked in US agriculture, current smoking status (yes/no), region of Mexico, and pack–years. The least square means and confidence intervals are reported after back-transformations. These data analyses were performed using SAS 9.1 (SAS Institute, Cary, NC) and p-values of less than 0.05 were considered statistically significant.