Exposure to Regular Gasoline and Ethanol Oxyfuel during Refueling in Alaska Lorraine C. Backer,1 Grace M. Egeland,2 David L. Ashley,1 Nicholas J. Lawryk,3 Clifford P. Weisel3 Mary C. White, Tim Bundy,4 Eric Shortt,5 John P. Middaugh2 1National Center for Environmental Health, Centers for Disease Control and Prevention, Atlanta, GA 30341-3724 USA; 2Epidemiology Section, Alaska Department of Health and Social Services, Anchorage, AK 99524-0249 USA; 3Environmental and Occupational Health Sciences Institute, Piscataway, NJ 08855-1179 USA; 4Alaska Department of Labor, Kenai, AK 99611 USA; 5State of Alaska Department of Labor, Anchorage, AK 99510-7022 USA Gasoline and other fuels contain toxic chemicals such as benzene that can cause a wide range of adverse health effects, includ- ing cancer (1). It has been estimated that more than 100 million people in the United States are briefly exposed to low lev- els of volatile organic compounds (VOCs) from gasoline during refueling activities (2). The major sources of exposure to gasoline during refueling are air that is displaced from the gasoline tank into the environ- ment when the tank is filled and vapors from accidental gasoline spills. Despite widespread exposure, very few studies have examined either exposure to gasoline during refueling or the possible health effects asso- ciated with these exposures. National ambient air quality standards (NAAQS) for outdoor air have been devel- oped by the EPA for six important air pol- lutants including CO (3). For CO, EPA has set a time-weighted average level of 10.3 mg/m3 (9 ppm) averaged over 8 hr as the national standard. The NAAQS level was exceeded in Fairbanks and Anchorage dur- ing 1991 and 1992; therefore, the state was required to introduce an oxygenated fuel program for the winter of 1992-1993. In October of 1992, gasoline containing 15% (by volume) methyl tertiary butyl ether (MTBE) as the oxygenate was introduced in Alaska. Shortly thereafter, the Alaska Department of Health and Social Services received numerous health complaints, report- edly due to exposure to the new gasoline for- mulation (e.g., when pumping gasoline), and a study of occupational exposure to the oxy- fuel was conducted by Moolenaar et al. (4). By December 1992, the governor of Alaska had suspended the oxyfuels program. During the winter of 1994-1995, the Alaska Department of Environmental Conservation (ADEC) introduced a CO emissions reduction program in Anchorage that included the use of the oxyfuel E- 10, a fuel made by mixing regular unleaded gasoline with ethanol to create a blend that is 10% ethanol by volume. Because E-10 had not been adequately tested under the environmental conditions typical of Alaskan winters, a wintertime engine per- formance study was initiated in Fairbanks. Stage II vapor recovery systems, which collect gasoline vapors at the pump nozzle and recirculate them into the underground gasoline storage tanks, were not in use in Alaska at the time the E- 10 oxyfuel was introduced. There was concern that the use of this fuel might increase the general pop- ulation's exposure to gasoline constituents, e.g., due to increased volatility of the E-10 blend. In response to this concern, and because of previous community complaints associated with the introduction of an oxy- fuel containing MTBE, we designed and implemented studies to characterize indi- vidual consumers' exposures to the aromatic components of regular gasoline while refu- eling and to examine whether the exposures changed when E-10 was introduced. We examined environmental (ambient air) exposure levels and internal levels (in blood) of several important constituents of gasoline in participants in Fairbanks, Alaska. Methods Recruitment of study participants. The pro- tocol was approved by the Institutional Review Board of the Centers for Disease Control and Prevention, and informed con- sent was obtained from all study partici- pants after the nature and possible conse- quences of the study were explained. The ADEC recruited 264 participants for its wintertime engine performance study through advertisements in newspapers and at gasoline stations. Participants in the study were blinded to the type of gasoline (regular or E-10) they were using by being assigned to purchase gasoline from either pump A or pump B, from approximately January through March 1995. Each time they pur- chased gasoline, participants completed a brief questionnaire on how their car was function- ing. When individuals volunteered to partici- pate in the wintertime engine performance study, they were also asked if they would be willing to participate in the Fairbanks gasoline exposure assessment. The ADEC compiled a Address correspondence to L.C. Backer, National Center for Environmental Health, Centers for Disease Control and Prevention, 4770 Buford Hwy NE, MS F46, Atlanta, GA 30341-3724 USA. The current address for M.C. White is Agency for Toxic Substances and Disease Registry, Atlanta, GA 30333 USA. The authors would like to acknowledge the many individuals who contributed to the success of this project: F. Cardinali and J. McCraw analyzed the blood samples; J. Roche obtained the blood samples; and S. Ryan, J. Weymiller, and D. Sweet assisted in conducting this study. This work was supported in part by Interagency Agreement #DW75937178-01 between the Centers for Disease Control and Prevention and the EPA. Received 13 November 1996; accepted 7 May 1997. Volume 105, Number 8, August 1997 * Environmental Health Perspectives 850 Articles * Exposure to gasoline and oxyfuel while refueling list of names and phone numbers of those interested in participating in our study. The staff from the Epidemiology Section of the Alaska Department of Health and Social Services contacted persons on this list until they had identified and recruited 30 people who had been using pump A and 30 who had been using pump B. To be eligible, peo- ple had to be nonsmokers who were not occupationally exposed to VOCs; 68 people were contacted before these target recruit- ment numbers were achieved. The people who declined to participate cited lack of time and concern about donating blood as reasons for not participating. On the scheduled date, each participant provided a 10-ml blood sam- ple, pumped gasoline, and then provided a second 10-ml blood sample and answered the questionnaire. To compensate them for their time, we provided study participants with a voucher worth $25 toward future gasoline purchases at the gasoline station where the study was conducted. Questionnaire. The questionnaire was designed to examine the frequency of self- reported symptoms (i.e., irritated eyes, headache, nausea, burning sensation in the nose or throat, cough, or dizziness) possibly associated with exposure to gasoline. Also included were questions regarding partici- pants' demographic characteristics, smoking status, and exposure to environmental tobacco smoke and whether, during the previous week, they had other illnesses (e.g., the flu or a cold). Participants' exposure to gasoline was assessed by asking them how many times in the last week they had pumped gasoline and how much time they spend in a motor vehicle on a weekday. The number of gallons of gasoline they pumped just prior to providing a blood sample and the number of seconds they spent pumping gasoline were also recorded. Biological measurements of internal dose for selected VOCs. VOCs have a very short half-life in blood. Because of the short half-life and because we wanted to avoid confounding exposures from envi- ronmental tobacco smoke in the gasoline station office, we arranged to have a recre- ational vehicle (RV) on site. We collected specimens from study participants in the RV immediately before and within 10 min after they finished pumping gasoline. Venous blood samples were collected in Vacutainer (Becton Dickinson, Rutherford, NJ) tubes containing sodium oxalate and sodium fluoride according to the method of Ashley et al. (5). Blood samples were kept cold and shipped to the National Center for Environmental Health in Atlanta, Georgia, within 48 hr of collection. Blinded blood samples were analyzed for a series of VOCs by purge and trap gas chromatography-mass spectrometry (GC- MS). The method is applicable to the deter- mination of VOCs in whole blood at extremely low detection limits and is described by Ashley et al. (5). Measurements were completed within 10 weeks of collec- tion in order to minimize any changes in blood VOC concentrations during refriger- ated storage. Detection limits for the analytes report- ed in this study are generally in the low parts per trillion (ppt) range. Gasoline con- stituents that were measured in blood, and their corresponding analytical limits of detection (LODs; in ppb), included ben- zene, 0.040; ethylbenzene, 0.024; m-lp- xylene, 0.055; o-xylene, 0.020; and toluene, 0.040. The concentrations of a series of VOCs not found in gasoline and thus not likely to change with a person's exposure to gasoline were determined and reported as part of quality assurance and quality control protocols. These additional VOCs and their corresponding LODs (in ppb) were 1,4-dichlorobenzene, 0.040; chloroform, 0.010; and styrene, 0.010. In addition to measuring the levels of gasoline constituents and the other VOCs listed above, we verified that all study par- ticipants were nonsmokers by measuring the levels of 2,5-dimethylfuran in their blood samples (6). Levels of 2,5-dimethyl- furan were below the detection limit of 0.024 ppb for all study participants, con- firming that none were active smokers. Some samples contained levels of one or more of the VOCs of interest that were below the analytical LOD. For values below the LOD, the values produced by the ana- lytical method were used in the analyses (7). Personal breathing zone samples. Personal breathing zone (PBZ) samples were collected according to the method described by Lioy et al. (8). SKC model 222-3 Low Flow Personal Air Samplers (SKC, Eighty Four, PA) equipped with sorbent tubes that contained a layered absorbant [0.ig Tenax GC (Alltech Corporation, Deerfield, IL), 0.1 g Carboxen 569, and 0.1 g carbosieve SIII (Supelco, Inc., Bellefonte, PA)] were used to collect the samples. The air-sampling pumps were placed in a pocket of a vest, which study participants put on over their clothing. The collection media were taped to the shoulder of the vest so that they would be in the participants' breathing zone. The sam- pling pumps were turned on when a partici- pant began pumping gasoline and were turned off immediately after they finished. Because PBZ VOC concentrations were assumed to be low, but were quantita- tively unknown, two simultaneous samples with different air volumes were collected for each individual. The air flow rate for one of each pair of samplers was set at 1 1/min, and the rate for the other sampler was set at 0.1 1/min. The air samples were analyzed by GC-MS within 2 weeks of col- lection. The method's analytical detection limits were 2 ng for toluene and m-lp- xylene and 5 ng for benzene, ethylbenzene, and o-xylene. Statistical analyses. Differences in base- line levels of each VOC in the blood of study participants when they arrived at the study site were accounted for by subtract- ing the level of each chemical in the partici- pants' blood before pumping gasoline from the level of each chemical in their blood after pumping gasoline. Neither the levels of VOCs in partici- pants' blood before pumping gasoline, the levels in their blood after pumping gasoline, nor the differences between the two were normally distributed; therefore, nonpara- metric methods were used to analyze the data. The Wilcoxon signed rank test (9) was used to compare participants' paired blood levels of VOCs before and after pumping gasoline, and the Wilcoxon two-sample test (9) was used to compare the changes in blood VOC levels among people who pumped regular gasoline with the change among those who pumped E-10. For the PBZ samples, analytical results from the two pumps running at different rates were averaged to produce a single value for each gasoline constituent for each partic- ipant. The data for the PBZ air samples were also not normally distributed; thus, we used the Wilcoxon two-sample test to com- pare the levels of VOCs in the PBZ air of those who pumped regular gasoline with the levels of VOCs in the PBZ air of those who pumped the E-10 blend. Spearman rank correlation coefficients were calculated using levels of gasoline constituents in blood and PBZ air for each person. Regression analyses were used to exam- ine confounders in the analysis of the asso- ciation between levels of VOCs in blood and ambient air and the type of gasoline pumped. Because the blood VOC data were not normally distributed and because the difference (after pumping gasoline minus before pumping gasoline) variable was sometimes negative, the difference was expressed as a ratio (after pumping/before pumping). The logl0 transformations of this ratio and the PBZ air data were used in regression analyses. We considered a vari- able to be a confounder if it changed the value for the parameter estimate for the type of gasoline pumped by 10% or more. The number of gallons of gasoline pumped was a statistically significant pre- dictor of exposure. Therefore, general linear models programs (10) were used to create Environmental Health Perspectives * Volume 105, Number 8, August 1997 851 Articles * Backer et al. least square mean values for exposure adjusted for the number of gallons pumped. Statistical analyses were performed by using SAS 6.10 for Windows (SAS Institute, Cary, NC) unless specified otherwise. Results Questionnaire. The demographic character- istics of the group that pumped regular gasoline were similar to those of the group that pumped the 10% ethanol blend (E-10). The average age was 45 years for both groups. There were 11 (36.7%) females in the group that pumped regular gasoline and 14 (46.7%) in the group that pumped E-10. One difference was in the amount of gasoline pumped the day of the study: peo- ple who used E-10 pumped somewhat more gasoline (mean 14.3 gallons) than those who pumped regular gasoline (mean 10.8 gallons; t-test, p = 0.08). One (3.3%) of the people who pumped regular gasoline and 3 (10%) of the people who pumped E- 10 reported spilling some gasoline when refueling on the day of the study. People in both groups reported very few of the symptoms likely to result from expo- sure to gasoline, and the prevalences of these symptoms were similar for both groups. For example, headache in the last week was the most commonly reported symptom, and it was reported by 7 (23.3%) of the people who pumped regular gasoline and 8 (26.7%) of the people who pumped E-10. VOCs in blood. Although measured lev- els of VOCs in a few samples were below the analytical LOD, we used the values calculat- ed by the method of Ashley et al. (5) in our analyses. This use of VOC measurements below the LOD was unlikely to affect our analyses or conclusions; benzene levels in blood were below the LOD in only one indi- vidual before pumping regular gasoline and in one individual before pumping E-10, and the levels of ethylbenzene were below the LOD for three individuals before pumping regular gasoline and for five individuals before pumping E-10. All reported values for m/p-xylene, o-xylene, and toluene were mea- surable levels (>LOD). The results from the analysis of gasoline constituents in whole blood are presented in Tables 1 and 2 and Figures 1 and 2. The results from a few blood samples were not reported either because the sample volume collected was insufficient for analysis (the study was conducted in extremely cold weather, it was cold inside our RV, and we sometimes were unable to obtain enough blood for the post-pumping sample) or because quality assurance/quality control requirements were not met when the sam- ples were analyzed. The levels of gasoline constituents in blood of participants before and after they pumped gasoline are pre- sented in Table 1 and Figures 1 (benzene) and 2 (toluene). Participants had signifi- cantly higher blood levels of gasoline con- stituents (p<0.05) after pumping gasoline than before pumping gasoline whether they pumped regular gasoline or E-10. For example, participants' median benzene concentration in whole blood increased from 0.19 ppb to 0.54 ppb (Wilcoxon Signed Rank Statistic, p<0.01) after pump- ing regular gasoline. Study participants' levels of the other VOCs not found in gasoline either did not change as a result of exposure to gasoline during refueling (1,4- dichlorobenzene and styrene) or decreased during refueling activities (chloroform). Levels of 2,5-dimethylfuran were below the detection limit of 0.024 ppb for all study participants, confirming that none were active smokers. Table 2 shows the differences in blood VOC concentrations (blood concentrations after pumping minus blood concentrations before pumping) for the group that pumped regular gasoline and for the group that pumped E-10. Blood benzene levels were somewhat higher for the group that pumped E- 10, but the differences in the internal doses (as measured by levels of gasoline con- stituents in blood) received by the two groups were not statistically significant. There were several potential con- founders that may have affected the associ- ation between the type of gasoline pumped and levels of VOCs in participants' blood or in their PBZ air. In the regression analy- sis in which the ratio measure of after- pumping values to the before-pumping val- ues (log-transformed) was the outcome measure, the initial model included as pre- dictors the type of gasoline pumped, the day of the study, and whether the person spilled gasoline that day. The type of gaso- line pumped had no statistically significant relationship with either benzene (p = 0.13) or toluene (p = 0.34) blood levels. A variable indicating whether the person had spilled gasoline was subsequently removed from all models because it was not statistically sig- nificant and its presence did not affect the parameter estimates for the other variables. The number of gallons of gasoline pumped Table 1. Blood volatile organic compound concentrations among participants before and after pumping gasoline, by type of gasoline, in February 1995 Regular gasoline (n = 26)b Ethanol blend (n = 22) Before pumping After pumping Wilcoxon Before pumping After pumping Wilcoxon median (range) median (range) signed rank median (range) median (range) signed rank Chemicala in ppb in ppb p in ppb in ppb p Constituents in gasoline Benzene 0.19 0.54 <0.01 0.18 0.70 <0.01 (0.040) (0.08-0.65) (0.13-1.70) (0.06-0.55) (0.14-4.20) Ethylbenzene 0.10 0.16 <0.01 0.11 0.16 <0.01 (0.024) (0.02-0.73) (0.06-1.40) (0.04-0.55) (0.06-0.64) m-/p-Xylene 0.44 0.58 <0.01 0.48 0.62 0.02 (0.055) (0.18-2.60) (0.31-4.90) (0.23-2.10) (0.25-2.50) o-Xylene 0.20 0.28 <0.01 0.26 0.30 <0.01 (0.020) (0.10-0.59) (0.15-1.50) (0.09-0.62) (0.15-0.72) Toluene 0.38 0.74 <0.01 0.38 0.85 <0.01 (0.040) (0.11-0.78) (0.22-3.30) (0.13-1.20) (0.26-3.10) Not in gasoline 1,4 Dichlorobenzene 0.04 0.02 0.18 0.04 0.03 0.64 (0.040) (