Fiedler et al. stand on a stainless steel table. This conﬁgura- tion allowed exposure of two subjects at a time. The technician communicated separately with each subject through headphones and could view subjects at all times through a two-way window. For subject safety, an electrocardio- gram (ECG) signal was collected using dispos- able electrodes and sampled at 996 Hz through the Flexcomp Biomonitoring system 1.51 B (Thought Technology Ltd., Montreal, Quebec, Canada). This allowed continuous monitoring of heart rate during the exposure period. The composition and relative weight of the VOC mixture used were similar to those in previous indoor air studies (Kjaergaard et al. 1991; Molhave et al. 1986; Otto et al. 1992) and are reported in Table 1 (Fan et al. 2003). d-Limonene, the most frequently identiﬁed ter- pene in indoor air, was also added to the mix- ture. A ﬂask containing the liquid mixture of these 23 compounds was heated to 250°C by a hot plate, ﬂash evaporated, and injected into the clean air stream that delivered the VOC mixture into the CEF. Constant concentrations of chemical compounds can be maintained in the CEF by injection of the chemicals continu- ously into the air supply, which ﬂows through the CEF without recirculation. The desired concentration of VOCs in the indoor environ- ment was achieved by adjusting the ﬂow rate of the delivering air using a mass ﬂow controller. Total VOC concentration in the air of the indoor environment was approximately 26 mg/m3, with the concentration of each compound below the threshold limit value recommended for occupational exposure by the American Conference of Governmental Industrial Hygienists (1992). MCA was Table 1. The mixture of VOCs. No. Compound Relative Concentration weight (mg/m3) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 Total 1544 n-Butylacetate p-Xylene n-Butanol n-Decane 1-Decene 1,1-Dichloroethane d-Limonene Ethylbenzene Ethoxyethylacetate n-Hexanal n-Hexane n-Nonane α-Pinene 2-Butanone Cyclohexane 3-Methyl-2-butanone 4-Methyl-2-pentanone n-Pentanal Isopropanol n-Propylbenzene 1,2,4-Trimethylbenzene n-Undecane 1-Octene 10 10 1 1 1 1 1 1 1 1 1 1 1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.01 8.25 8.25 0.825 0.825 0.825 0.825 0.825 0.825 0.825 0.825 0.825 0.825 0.825 0.083 0.083 0.083 0.083 0.083 0.083 0.083 0.083 0.083 0.008 26.330 End 180 Relax 170 Guess exposure condition 5-min postexposure—clinical center: - Lung function - Sym7 - Neuro4 - Nasal lavage generated by introducing a 1-min spike of the VOC mixture at approximately 10% of the exposure concentration for the VOC exposure condition. The maximum total VOC concen- tration in the CEF during the MCA condition was approximately 2.5 mg/m3. Given the air exchange rate used in the experiments, it took about 90 min for the masking concentration to decay to 0.25 mg/m3. The background O3 concentration is < 3 ppb in the CEF. O3 generated in situ by an O3 generator was delivered into the CEF and was maintained at a steady-state concen- tration of 40 ppb. This was consistent with the intention to examine the effect of products of O3–VOC reactions rather than O3 itself. Table 2 compares the concentrations of by- products generated in the VOC + O3 condi- tion relative to the VOC condition. Exposure generation and characterization were described in detail in a previous publication (Fan et al. 2003). Procedures Before receiving a complete physical examina- tion, including a medical history review and routine blood chemistries, subjects who met inclusion criteria gave written informed consent according to the University of Medicine and Dentistry of New Jersey–Robert Wood Johnson Medical School Institutional Review Board. To reduce practice and novelty effects, subjects were shown the CEF and trained to perform the fol- lowing procedures: symptom questionnaire, speech and neurobehavioral tasks, salivary sam- pling for cortisol and estradiol, nasal lavage, and spirometry. Subjects were randomly assigned to the order of exposure conditions, half with and half without the stressor. Subjects were told dur- ing informed consent that they may be asked to perform a public speaking task. They were not told whether they would be performing the task before any exposure session. Thus, subjects were blind to the stress condition. Neither the subject nor the research technicians were told exposure conditions. Each experimental session was 3 hr in dura- tion and occurred in the morning. Subjects were asked not to use caffeine or alcohol on the day before and on the day of the testing session. Subjects also could not have an active upper res- piratory illness (either infection or allergy) or use medication for allergies or other respiratory conditions for 1-week before each exposure Table 2. Summary of products observed during different exposure conditions. Exposure conditions MCA VOCs (23 VOCs) VOCs + O3 (O3 + 23 VOCs) Ultraﬁne particle number concentration (particles/cm3)a NAd 2,500 46,000 Particle mass concentration Formaldehyde p-Tolualdehyde (µg/m3)b (µg/m3)b (µg/m3)b < 5 3–7 140 7 13 40 ND ND 6.2 Glyoxal (µg/m3)b ND ND 4.6 Hydrogen peroxide (ppb)a,c NAd 0.3 1.9 ND, not detected. aTwo-hour average concentration. bFour-hour average concentration. cIncludes hydrogen peroxide and organic hydroperoxides. dNot measured but expected to be the same or lower than the 23 VOC-only condition. Figure 1. Time line for experimental procedure. Abbreviations: Cort1, cortisol collection at minute 15; Cort2, cortisol collection at minute 90; Cort3, cortisol collection at minute 110; Cort 4, cortisol collection at minute 130; Neuro1, neurobehavioral task (POL) at minute 15; Neuro2, Neurobehavioral task (POL) at minute 60; Neuro3, neurobehavioral task (POL) at minute 140; Neuro4, neurobehavioral task (POL) at 5 min postexposure; Sym1, symptom questionnaire at baseline; Sym2, symptom questionnaire at minute 15; Sym3, symptom questionnaire at minute 60; Sym4, symptom questionnaire at minute 90; Sym5, symptom questionnaire at minute 110; Sym6, symptom questionnaire at minute 130; Sym7, symptom questionnaire at 5 min postexposure. VOLUME 113 | NUMBER 11 | November 2005 • Environmental Health Perspectives Baseline: 30 min before exposure session—clinical center - Lung function - Estradiol - Nasal lavage - Sym1 Relax Minute 1 10 Subject Sym2 enters cort 1 CEF neuro 1 Begin Baseline physiology Exposure 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 Sym3 neuro 2 Vigilance Sym4 Stress/ Sym5 Typing Sym6 Neuro3 cort 2 math cort 3 cort 4