Residential air pollution and infant mental development affects mental development during the sec- ond year of life, and whether antioxidant and detoxification factors modulate this associa- tion, in a population-based birth cohort in which air pollution exposures were not associ- ated with social class determinants (Vrijheid et al. 2010). Materials and Methods Study design and participants. Population- based birth cohorts were established as part of the INMA (INfancia y Medio Ambiente; Environment and Childhood) Project in sev- eral regions of Spain following a common protocol (Guxens et al. 2011). This analysis uses the INMA cohorts of Valencia, Sabadell (Catalonia), Asturias, and Gipuzkoa (Basque Country) established between 2003 and 2008. Pregnant women were enrolled during the first trimester of pregnancy at public pri- mary health care centers or public hospitals, depending on the region, providing they ful- filled the inclusion criteria (≥ 16 years of age, intention to deliver at the reference hospital, no problems of communication, singleton pregnancy, no assisted conception); 99.5% of Spaniards have public health insurance, and 70–90% of women use public health services during pregnancy (Regidor et al. 2008). Of all women invited, 56% agreed to partici- pate. Women were then followed throughout pregnancy. Their children were followed from birth through 2 years of age. Informed con- sent was obtained from all participants, and the study was approved by the hospital ethics committees in each participating region. Child mental development test. Children’s mental development was assessed at around 14 months of age (range, 11–23 months) using the Bayley Scales of Infant Development (Bayley 1993). The Bayley mental develop- ment scale consists of 163 items that assess age-appropriate mental development, includ- ing performance abilities, memory, and early language skills. All testing was done in the health care center in the presence of the mother, by 12 specially trained psychologists. Psychologists were not aware of any exposure information. To limit interobserver variability, we applied a strict protocol, including training sessions where interobserver differences were quantified and three sets of quality controls (interobserver reliability tests) undertaken dur- ing the fieldwork. The interrater reliability, estimated by intraclass correlation, was 0.90, and Cronbach’s alpha coefficient, a measure of internal consistency, was 0.70. Tests were per- formed on 2,213 children who attended the visit. Eighteen children were excluded because of specific pathologies, and 144 were excluded because their test results were of uncertain quality due to less than optimal cooperation. Raw scores were standardized for child’s age in days at test administration using a parametric method for the estimation of age-specific refer- ence intervals. The parameters of the distribu- tion were modeled as a fractional polynomial function of age and estimated by maximum likelihood. Residuals were then standardized to a mean of 100 points with a standard devia- tion of 15 points to homogenize the scales. Assessment of air pollution exposure. A complete description of the methodology on exposure modeling has been reported previ- ously (Aguilera et al. 2008; Estarlich et al. 2011; Fernández-Somoano et al. 2011; Iñiguez et al. 2009). Briefly, ambient con- centrations of nitrogen dioxide (NO2) and benzene were repeatedly measured with pas- sive samplers distributed over the study areas according to geographic criteria, taking into account the expected pollution gradients and the expected number of births. The sam- plers measured pollutants levels using radial symmetry (Radiello®; Fundazione Salvatore Maugeri, Padua, Italy) and remained exposed during various sampling periods of 7 days each. Land use regression models were used to predict NO2 and benzene levels at wom- en’s residential addresses, taking into account any residential changes during pregnancy. Geographic information system data derived in ArcGIS version 9.1 (ESRI, Redlands, CA, USA) (traffic as measured by vehicle density, distance of the home from a main road, land use, and altitude) were used to obtain pre- dictor variables. Spatial estimates were tem- porally adjusted using serial records from the network of monitoring stations covering the study areas, to obtain estimates for each woman’s specific pregnancy period. Finally, an average exposure level over the whole preg- nancy period was calculated. Antioxidant/detoxification variables. Information on maternal diet was obtained in the first trimester of pregnancy using a 101-item semiquantitative validated food fre- quency questionnaire (Vioque et al. 2007). Women were asked to report usual intakes since the start of pregnancy using refer- ence portion sizes and nine frequency cat- egories ranging from “never/less than once per month” to “6 or more times per day.” The food frequency questionnaire included 10 fruit items (oranges; fresh-squeezed orange juice; bananas; apples or pears; peaches, nec- tarines, or apricots; watermelon or melon; grapes; plums; kiwis; and olives) and 12 veg- etable items (spinach; cabbage, cauliflower, or broccoli; lettuce or endive; tomatoes; onions; carrots or squash; green beans; eggplant or zucchini; bell peppers; artichokes; asparagus; and garlic). Reported intakes of each item were converted to estimated daily frequencies using the midpoint of each category (e.g., a frequency of 1–3 times/month was converted to 2 times/30 days or 0.067 times/day). All items were summed to estimate overall fruit Environmental Health Perspectives • volume 120 | number 1 | January 2012 and vegetables intakes. Fruit and vegetable intakes were categorized in two groups: low tertile (≤ 405 g/day) versus medium/high ter- tile (> 405 g/day). Detailed information about child feeding through the second year of life was collected from mothers by interviewer-administered questionnaires. Breast-feeding was defined as receiving any breast milk, regardless of supplementation with food or other liq- uids, including nonhuman milk. Duration of breast-feeding was categorized into three groups: children who were never breast-fed, children who were breast-fed for a short time period (< 6 months), and long-term breast-fed children (≥ 6 months). Plasma 25-hydroxyvitamin D3 [25(OH)D] reflects contributions from all sources of vita- min D (i.e., diet and sun exposure) and is considered the best circulating biomarker of vitamin D metabolic status. A single mater- nal fasting blood specimen was drawn dur- ing pregnancy (mean ± SD, 13.4 ± 1.7 weeks of gestation). Samples were processed imme- diately and stored at –70°C to –80°C until analysis. Levels of maternal plasma vitamin D were determined by high-performance liq- uid chromatography using a BioRAD (Madrid, Spain) kit according to Clinical and Laboratory Standard Institute (NCCLS) pro- tocols. Because 25(OH)D levels are known to vary over the course of the year, we used season-specific cut points in the analyses to deal with season of blood drawn (spring, sum- mer, fall, and winter). Season-specific tertiles were constructed: low (< 22.1, < 30.7, < 25.2, and < 21.0 ng/mL for spring, summer, fall, and winter, respectively), medium (22.1–32.0, 30.7–39.3, 25.2–33.7, and 21.0–30.7 ng/mL, respectively), and high (> 32.0, > 39.3, > 33.7, and > 30.7 ng/mL, respectively). Other parental and child variables. Questionnaires during the first trimester of pregnancy obtained information about the highest achieved level of parental education, parental occupation, parental country of ori- gin, parental age, maternal height and pre- pregnancy weight, parity, and marital status. We defined parental social class from the maternal or paternal occupation during preg- nancy based on the highest social class, using a widely used Spanish adaptation of the inter- national ISCO88 (International Standard Classification of Occupations) coding sys- tem (Instituto Nacional de Estadística 1994). Maternal tobacco and alcohol use, second- hand smoke at home or in the workplace, use of a gas stove at home during pregnancy, and annoyance due to noise at home were collected through questionnaires during the third trimester. Maternal levels of hemoglo- bin and thyroid-stimulating hormone were analyzed in maternal serum extracted in the first trimester. Maternal cotinine levels 145