Weekly Personal Ozone Exposure and Respiratory Health in a Panel of Greek Schoolchildren

Background: The association of ozone exposure with respiratory outcomes has been investigated in epidemiologic studies mainly including asthmatic children. The findings reported had methodological gaps and inconsistencies. Objectives: We aimed to investigate effects of personal ozone exposure on various respiratory outcomes in school-age children generally representative of the population during their normal activities. Methods: We conducted a panel study in a representative sample of school-age children in the two major cities of Greece, Athens and Thessaloniki. We followed 188, 10- to 11-y-old, elementary school students for 5 wk spread throughout the 2013–2014 academic year, during which ozone was measured using personal samplers. At the end of each study week, spirometry was performed by trained physicians, and the fractional concentration of nitric oxide in exhaled air (FeNO) was measured. Students kept a daily time–activity–symptom diary and measured PEF (peak expiratory flow) using peak flow meters. Mixed models accounting for repeated measurements were applied. Results: An increase of 10 μg/m3 in weekly ozone concentration was associated with a decrease in FVC (forced vital capacity) and FEV1 (forced expiratory volume in 1 s) of 0.03L [95% confidence interval (CI): −0.05, −0.01] and 0.01L (95% CI: −0.03, 0.003) respectively. The same increase in exposure was associated with a 11.10% (95% CI: 4.23, 18.43) increase in FeNO and 19% (95% CI: −0.53, 42.75) increase in days with any symptom. The effect estimates were robust to PM10 adjustment. No inverse association was found between ozone exposure and PEF. Conclusions: The study provides evidence that airway inflammation and the frequency of respiratory symptoms increase, whereas lung function decreases with increased ozone exposure in schoolchildren. https://doi.org/10.1289/EHP635


Introduction
Ozone (O 3 ), a very reactive gas and strong oxidant, is found as a secondary pollutant in the troposphere. Although, its presence in the stratosphere is essential for life because it filters harmful ultraviolet radiation, increased concentrations of O 3 in the air we breathe have been linked to adverse health effects mainly concerning the respiratory system (WHO 2013). A number of controlled human exposure studies have demonstrated changes in FEV 1 and respiratory symptoms (Adams 2006;Horstman et al. 1990; Kim et al. 2011) as well as lung inflammation (Devlin et al. 1991;Kim et al. 2011), but those included healthy adults, not children. In an attempt to investigate the acute effects of ozone under real-world exposures several camp studies have been conducted involving sequential measurements of lung function and ambient ozone measurements on children attending summer camps (Burnett et al.1990;Higgins et al. 1990;Raizenne et al.1989;Spektor et al. 1988;Spektor et al. 1991). However, despite the evidence they provide of an association between daily outdoor O 3 concentrations and decreased lung function (Kinney et al. 1996), the lack of personal measurements, variations in data reporting and analysis as well as concerns about potential confounding by other pollutants that may co-vary with O 3 , limit their use in risk assessment. Therefore, so far there is lack of studies conducted in children under real living conditions, at current concentrations of ozone, using personal exposure assessment.
Children's lungs are considered to be more sensitive and vulnerable to air pollution as they have, even at rest, a larger surface area for body size compared with adults and are still growing. Children breathe more air per unit of body weight and have different breathing patterns than adults that alter the deposition and toxicity of inhaled gases and particles (Arcus-Arth and Blaisdell 2007; Bateson and Schwartz 2008;Warren et al. 1990). Moreover, because they spend more time outdoors and are more active, they have higher ventilation rates leading to increased intake and deposition of pollutants (Bateson and Schwartz 2008). So far, epidemiological studies on the effects of ozone on children's lung function and respiratory symptoms have mainly focused on asthmatics and have reported inconsistent results (Castro et al. 2009;Declercq and Macquet 2000;Jalaludin et al. 2000;Just et al. 2002;Li et al. 2012;Mortimer et al. 2002;Scarlett et al. 1996). However, response to ozone exposure displays individual variability that cannot be explained by underlying morbidity (Schelegle et al. 2012;WHO 2013), and thus, from a public health point of view, it is critical to determine to what extent ambient O 3 exposure is responsible for adverse respiratory effects in the general population of children.
We conducted a panel study (Respiratory Effects of Ozone Exposure in children; RESPOZE) in a representative sample of the general population of schoolchildren in the two major cities of Greece, Athens (state capital) and Thessaloniki. Greece provides a unique opportunity to study O 3 health effects because of its sunny, warm weather as well as the high concentrations of precursor pollutant emissions encountered in some of its cities.
In this paper we examine the association of weekly personal the Mini-Wright peak flow meter three times every morning, ozone measurements with lung function parameters, airway afternoon, and night before the use of any medication. In addiinflammation, and respiratory symptoms. tion, at the end of each day they were asked to record all their daily symptoms, namely cough, wheezing, breathing difficulties

Materials and Methods
(dyspnea), fever, and stuffy nose; school absenteeism; and daily medication use (yes vs. no, as well as identification of medicine).

Study Design: Subjects
A panel study was conducted, in the two largest and most pol-Spirometry luted cities of Greece, Athens and Thessaloniki (total population Spirometry was performed at school at the end of each of the five of about 4 million). The research was approved by the Ethics intensive field work weeks. Thus, a total of five spirometric Committee of the National and Kapodistrian University of assessments were available per schoolchild. All spirometry Athens, and it complied with all relevant national and local regumaneuvers were performed by the same pulmonologists or by the lations. The study population consisted of all children in the fifth specially trained pediatrician in a separate office provided by grade (age 10-11 y) in state elementary schools representative of each school that was quiet, well ventilated and had a standard the general population of students in that age group. State schools temperature independent of season. Children were tested in a were chosen because all attending students are required to live in seated position and with nose clips in place. Portable spirometers the school neighborhood. A two-stage sampling design was (Spiropalm, Cosmed Srl, Italy) compatible with ATS (American applied. First, state elementary schools located near a fixed moni-Thoracic Society) and ERS (European Respiratory Society) toring site (within 2 km), were identified, and permission to requirements were used (Miller et al. 2005). At least two techniimplement the project was obtained from the Ministry of cally satisfactory maneuvers had to be performed with a differ-Education. In the second stage, the research team visited all ence between the two highest values of FVC (forced vital schools and informed fifth grade school children in class about capacity) and FEV 1 of <5% of each other. The best FVC and the project. Informed consent was obtained from the parents of FEV 1 were recorded independently of the order of the maneuver, children willing to participate and the final sample was formed.
whereas the values for the other parameters were chosen from the We had estimated that a sample of 200 school children (100 from maneuver having the largest sum of FVC and FEV 1 (Miller et al. Athens and 100 from Thessaloniki) would provide power >90% 2005). PEF and FEF 25-75 were also recorded. to detect a decrease in FEV 1 (forced expiratory volume in 1 s) of 0.14% per 10 lg 3 =m in O 3 exposure, at the 5% significance level, F eNO considering that the overall proportion of variance explained A measurement of F was performed at school at the end of would be 41%. In each city 60% of the sample was drawn from eNO each field study week just before spirometry in the same room high-O 3 areas (suburbs), to ensure that we had enough relatively where spirometry was performed (see above), thus, a total of highly exposed children, and 40% from low-O 3 areas (city cen-five fi measurements were available per schoolchild. The NIOX MINO ters, where ozone is scavenged by primary pollutants). All eld (Aerocrine, Solna, Sweden), an instrument that has also a scrubwork was implemented during the academic year 2013-2014 and ber to eliminate ambient NO from the subject s sample, was used consisted of five weeks of intensive follow-up per study partici-' for all measurements according to American Thoracic Society pant: two in the fall period, one in winter, two in spring/summer.
(ATS) and European Respiratory Society (ERS) recommenda-During each period, we used a staged entry of the participants for tions (ATS/ERS 2005). practical reasons. As a result, the period of data collection was longer, and thus, the likelihood for uncontrolled factors or unexpected events to influence the association between air pollution Ozone Exposure Assessment and health decreased (Roemer et al. 2000). Before the start of the To assess O 3 weekly exposure, each student was provided with a field work, trained interviewers visited the schoolchildren's famipersonal Ogawa O 3 sampler (Ogawa & Co. USA Inc., Pompano lies at home to fill in an extensive questionnaire with information Beach, FL) at the first visit of each field work week and was on demographic, life style, and residential characteristics and the instructed on how to wear and handle it. The sampler was colmedical history of the child. During this visit they also provided lected during the second visit at the end of the week. One sampler a peak flow meter (Mini-Wright, Clement Clarke International was placed in the outdoor space of each participating school also Ltd.) and trained the student on how to use it. A team of three providing a weekly measurement. Additionally, concentrations of field workers, including one pediatrician or pulmonologist, visdaily ambient O 3 as well as PM 10 (particulate matter with aerodyited each school twice for each field work week. At the first visit namic diameter ≤10 lm) and NO 2 (nitrogen dioxide) were an O 3 personal sampler (Ogawa USA) and a time-activity diary obtained by the nearest fixed monitoring site from the state net-(TAD) were given to the participating students. At the second work (http://www.ypeka.gr/). visit, a week later (same weekday), the O 3 personal sampler and completed TADS were collected, spirometry was performed, the Confounder Data fractional concentration of nitric oxide in exhaled air (F eNO ) was A possible learning or fatigue e ect as well as measured and a 24-h dietary recall questionnaire was filled in by ff a growth factor throughout the academic year was taken into account by includinterview. [See "TAD-PEF (Peak Expiratory Flow): Symptoms"; ing a time trend as a variable taking values 1-5 for each consecu-"Spirometry"; "FeNO"; "Ozone Exposure Assessment"].
tive week (week ID). Additionally, all models were adjusted for sex, city (i.e., Thessaloniki vs. Athens) and area (high-vs. low-

TAD-PEF (Peak Expiratory Flow): Symptoms
ozone area, as used in the sampling frame), father's education Participating schoolchildren were instructed to complete a TAD (years) as socioeconomic index, outdoor ambient temperature collecting information on his or her location (at home, outdoors, ( C, weekly average and alternatively same day temperature), indoors, but not at home and in transport) at 15-min intervals for consumption of antioxidant foods (yes/no, in the corresponding each day of the field work period. In the TAD, they additionally 24-h recall), time spent outdoors (hours/day, from data recorded recorded a total of nine PEF recordings per day, performed with in the corresponding week TAD), and medication use (yes/no, during the corresponding week). Height (cm) and weight (kg) were adjusted in models with lung function outcomes. Models were additionally adjusted for PM 10 levels from the nearest fixed monitoring site.

Quality Assurance and Quality Control
Standard operating procedures (SOPs) were applied for air pollution and health measurements. A training workshop for all field workers was organized before the start of the fieldwork. It should be noted that especially for the TAD, which are demanding on the subject's time and accuracy, scrupulous data quality procedures were applied as well as controls with manual inspections of the data and cross references from the field workers' progress diaries.

Statistical Analyses
Random effects models, incorporating a random intercept, Stata Statistical Software (Release 12; StataCorp LP), were used to account for the repeated measurements, for each child. We applied multiple linear regression models when we investigated O 3 effects on continuous variables: PEF measurements from the Mini-Wright (taken as the average of seven daily values calculated as the mean of the three maximum values from the morning, afternoon, and evening measurements), spirometry indices FVC, FEV 1 , PEF, and FEF 25-75% (forced expiratory flow at 25-75%) as well as F eNO measurements, which were log-transformed because the distribution was skewed. Poisson models were used for counts: the number of days per week when any symptom occurred and the number of days when the child was absent from school. In all models, we adjusted for all the potential confounders mentioned above. Because ozone concentrations are higher in spring-summer, we repeated the analysis for this period only. In addition, because asthmatic children may present special characteristics, we repeated the analysis excluding this subgroup using two alternative definitions. The first definition was based on parents' reporting a doctor-diagnosed asthma. The second definition further included those students having FEV 1 =FVC and/or FEF 25 − 75% < LLN (lower limit of normal) at least once during the study period (provided that all spirometry maneuvers were performed according to the project's protocol), which provides evidence of obstructive ventilatory defect (Lougheed et al. 2012;Lum and Stocks 2010) and may denote (undiagnosed) asthma. We also conducted additional sensitivity analyses in healthy children only, excluding alternatively the two groups (asthmatic and asthmatic plus those presenting obstructive ventilatory defect) according to the definitions above.
Results are presented per 10-lg=m 3 increase in O 3 levels, representing approximately an interquartile range of the weekly measurements and thus, allowing comparability with previously published results based on measurements from fixed sites monitors. In sensitivity analyses we tested for heterogeneity between children, which included adding random slopes to our models.

Results
The final sample included 188 school-age children (97 in Athens and 91 in Thessaloniki). Table 1 shows personal, medical, socioeconomic characteristics, and respiratory health indices of the study population, by city and ozone concentration area (as used for the sampling scheme), during the academic year 2013-2014. Based on demographic data children residing in the two cities were similar. Parents reported a doctor-diagnosed asthma for 21 schoolchildren (7 in Athens and 14 in Thessaloniki). Of the remaining 167 children without a reported history of asthma, 22 (10 in Athens and 12 in Thessaloniki) had FEV 1 =FVC and/or FEF 25 − 75% < LLN (lower limit of normal) for at least once during the study period.
In Table 2 descriptive characteristics of the air pollutants and temperature by city and high-or low-O 3 area (as used in the sampling scheme) are presented. O 3 personal measurements were much lower than outdoor measurements at schools or fixed sites, reflecting the amount of time spent indoors, where ozone concentrations are lower. Furthermore, O 3 as well as PM 10 concentrations and temperature were higher in Athens than in Thessaloniki. Personal ozone and outdoor school ozone concentrations were in a range of 25-40% higher in high-versus low-ozone areas in both cities. In Athens, PM 10 concentrations were lower by 20% in highcompared with low-ozone areas but, in contrast, in Thessaloniki they were 5% higher in high-ozone compared with low-ozone areas. Therefore, in Athens there was a larger contrast in both O 3 and PM 10 concentrations between high-and low-ozone areas. In additional, contrasts were higher in outdoor school measurements compared with personal measurements.
Concerning the assessment of health effects, a negative association between weekly O 3 exposure and FVC was observed (both in the one and two pollutants models including PM 10 ). An increase of 10 lg=m 3 in weekly O 3 concentration was associated with a decrease in FVC of 0:03 L or 30 mL (95% CI: −0:05 L, −0:01 L). When we restricted the analyses to the spring-early summer period the negative effect of O 3 on FVC persisted. The results concerning the weekly O 3 effect on FEV 1 were similar. A 10-lg=m 3 increase in weekly O 3 concentration was associated with a decrease in FEV 1 of 0:01 L or 10 mL (95% CI: −0:03 L, 0:003 L). Peak expiratory flow, as assessed by spirometry, and FEF 25-75% , were not associated with weekly O 3 exposure in any period of analyses (Table 3). Weekly averages from daily values of PEF, taken with the Mini-Right flow meter, were positively associated with O 3 and this association remained when we restricted the analyses to the spring-early summer period (Table 3). Inclusion of PM 10 did not change the above-mentioned results.
Analysis of the association of F eNO with O 3 showed that a 10-lg=m 3 increase in weekly personal O 3 concentration was associated with an increase in F eNO values by 11.10% (95% CI: 4.23%, 18.43%). The direction of the effect and the magnitude of the association was practically the same after adjusting for PM 10 [9.5% (95% CI: 2.46%, 16.98%)] (Table 3). Associations with F eNO remained significant when we restricted the analysis in the springearly summer study period 11.79% (95% CI: 2.54%, 21.82%).
An increase of 19% (95% CI: −0:53%, 42.75%), (21%, 95% CI: 0.42%, 45.66%, after adjusting for PM 10 ) in the number of days with at least one symptom within each of the five weeks was associated with a 10-lg=m 3 increase in weekly ozone exposure (Table 4). The association was smaller and nonsignificant when only the spring-summer period was considered (data not shown). No association between weekly O 3 personal exposure with "any absenteeism" (i.e., the number of days the student was absent from school during each of the five weeks) was observed (Table 4). In sensitivity analyses, the direction of the effects remained unchanged when the groups of asthmatic, asthmatic plus those having obstructive ventilatory defect, or healthy children were analyzed separately. We observed no significant heterogeneity in the effects between children when we allowed for random slopes in our models (data not shown). Results remained unchanged when the same day temperature was included in the models. In addition, no effect modification by area (high or low ozone) or city (Athens or Thessaloniki) was detected.

Discussion
In this panel study, we found consistent associations between increased weekly O 3 personal exposure and a decrease mainly in Environmental Health Perspectives 077016-3 FVC and also FEV 1 , in 10-y-old school children following their normal daily schedules not modified by their participation in the study. We also observed corresponding increases in the number of days that any respiratory symptom occurred and in F eNO , a marker of airway inflammation. Our sample included asthmatic children (about 7%), but separate analyses by asthmatic and nonasthmatic children did not show a modification in the effect estimates. The students were sampled from low-and high-O 3 areas and from two major cities, but no effect modification by area or city was detected. Previous longitudinal studies investigating the effect of shortterm exposure to O 3 on children's respiratory health involved mainly asthmatic children and relied on fixed monitoring sites rather than on personal measurements ). Most studies used PEF as lung function parameter. Moreover, among the few panel studies concerning healthy children no one, to the best of our knowledge, combined inflammatory response and change in lung function with personal measurements of O 3 exposure Chen et al. 2015).
One very consistent and robust finding in our study is the increase in F eNO that persisted after controlling for PM 10 , probably reflecting the capacity of O 3 to induce airway inflammation (Mudway and Kelly 2000) even in healthy children. Very few studies investigated exposure to O 3 and F eNO levels. A panel study, involving school children living in Mexico City, demonstrated an association of F eNO with acute exposure to traffic-related air pollutants in both asthmatics and nonasthmatics . However, the 8-h moving average outdoor concentration of O 3 was associated with F eNO in asthmatic children only. Another study investigated the effects of exposure to several pollutants on F eNO in asthmatic children in the Mexican-U.S. border and found respiratory effects attributed to PM (several size fractions) and NO 2 but not of O 3 (Sarnat et al. 2012). Exposure assessment in these studies was based on fixed monitoring sites and passive samplers located in schools, resulting perhaps in less accurate assessment of O 3 exposure. Furthermore, we found significant negative associations, robust after adjusting for potential confounders and more strongly during warmer days, between O 3 weekly personal exposure and FVC as well as FEV 1 (but of borderline significance).
Based on our results, the mean decrease found in FVC (0:0 3L, per 10-lg=m 3 increase in personal O 3 exposure) may be of clinical significance if repeated exposures lead to a more permanent adverse effect. Moreover, the range of personal exposure measurements is about 40 lg=m 3 , depending not only on ambient concentrations but also on the child's time spent outdoors, and in the highest exposure range the weekly decrease in FVC may be clinically relevant. Concerning our findings on F eNO , it follows that a 20% increase in F eNO is associated with 20-lg=m 3 increase in O 3 personal exposure (an exposure contrast well within the range of measured exposures from 1 to 42 lg=m 3 ) considered to indicate an effect that may have clinical importance (Dweik et al. 2011).
a This definition of high-and low-concentration areas was based on previous years and used as a basis for the sampling procedure.  28:9 ± 7:4 23:1 ± 7:1 18:9 ± 3:8 21:0 ± 9:3 Temperature ( C) 17:6 ± 2:5 17:6 ± 2:5 14:6 ± 3:9 13:7 ± 3:8 Note: Data are presented as mean ± SD. The high-or low-O 3 concentration areas were used for the sampling scheme. with PEF were observed, with the largest study (at Pine Springs Ranch, east of Los Angeles) reporting a positive and statistically significant association (Kinney et al. 1996). In a Brazilian study, Castro et al. (2009)  Two panel studies examined the effect of O 3 exposure on FEV 1 in asthmatic children and found no effect (Dales et al. 2009;Delfino et al. 2004). Both of them relied on self-measured FEV 1 by handheld electronic devices. More consistent findings have been observed in the few studies using spirometry performed by trained personnel. In a reanalysis of six summer camp studies, a consistent decrease in FEV 1 has been found associated with increased ambient exposure to O 3 (Kinney et al, 1996). In the Mexican study ), using fixed site outdoor measurements, the investigators report negative associations with FVC and FEV 1 , although none reached the nominal level of significance. In a study in Taiwan (Chang et al. 2012), based also on fixed site measurements for the assessment of O 3 exposure, a significant negative association was observed with FVC and a nonsignificant negative association with FEV 1 . The decrements, we observed mainly in FVC and also in FEV 1 , are also consistent with previous experimental studies in young adults. The main reason for this O 3 -induced decrease is believed to be impaired inspiration as a result of stimulation of airway receptors causing a reduction in the level of inflation achieved at full inspiration (Blomberg et al. 1999;Hazucha et al. 1989;Kjaergaard et al. 2004). Based on all the above, our results support that ambient O 3 exposure affects inspiratory capacity and is related to airway inflammation.
In the present study, we also found a significant increase in respiratory symptoms after increased exposure to O 3 . This is a consistent finding among studies that investigate O 3 effects at different time scales, mostly in asthmatic children. Thus, Declerq and Macquet (2000), Delfino et al. (2002), Escamilla-Nuñez et al. (2008), Just et al. (2002), Mortimer et al. (2002), and Schlink et al. (2002) found an association of O 3 exposure and increase in respiratory symptoms, but few others found no effect (Jalaludin et al. 2004;Ostro et al. 2001). In our study asthmatic children were not found to be more susceptible than nonasthmatic children and this has been reported by other studies as well (Ward et al. 2002).
Our study has certain limitations. Our sample included children who agreed to participate from randomly selected schools; however, they did not form a random sample from the students of their class. This may have introduced some selection bias because children from more educated families or families more sensitive to environmental problems may have been preferentially included. However, we think that this did not bias our results away from the null given that the students attending a specific school are socioeconomically comparable (they live in the neighborhood) and the percentage of asthmatic children in our sample is comparable to that reported for the population (Papadopoulou Table 3. Mean change in lung function indices associated with weekly increase of 10 lg=m 3 in O 3 exposure measured by personal monitors in 178 school children.

Health outcome Models
O 3 effect per 10 lg=m 3 All study periods Spring-early summer period b-coefficient (95% CI) Wald   . Another limitation of our project is the fact that assessment of exposure to pollutants other than O 3 was not based on personal measurements but was estimated using the nearest fixed site monitors. This might have led to residual confounding, especially taking into account exposure to PM. We believe that the probability of residual confounding is limited because the correlations of O 3 with PM 10 and NO 2 was not the same across the high-and low-O 3 areas and between the two cities. Furthermore, PM is more homogeneously distributed and penetrates indoors making the fixed site and personal measurements less different from those of O 3 . Absenteeism might be a source of bias to the null for the estimated effects. During the field work, on each of the days our researchers visited the schools for spirometry, only a few children were absent from school (0-4 children each time, except during the last summer week in Thessaloniki when 17 children could not be found because specific schools had end-ofyear activities), and it may be hypothesized that some of these children might have had a respiratory problem because the reason for the absence was not reported by the school. Another limitation of our study is the inability to study children during the hottest months of the year, July and August, because of school holidays. These months are also characterized by high-O 3 concentrations. We were not able to reach schoolchildren during the holidays, but it should be mentioned that most families in Greece take their holidays during this time and the population of children in the cities is greatly reduced. An advantage of the present study is the use of personal O 3 monitoring. The higher contrast observed in outdoor school measurements compared with personal ones denotes the importance of relying on personal measurements when assessing O 3 exposure health effects on school children. Another advantage is the spirometry performed by physicians, which provides more accurate and valid results compared with self-monitoring. This may explain our findings associating ozone exposure with the weekly FVC and FEV 1 , provided through spirometry, whereas an analysis of daily PEF measured by peak flow meters, handled by the same sample of students, did not provide significant results (Samoli et al. 2017).
Among the strengths of our study is the study design, as panel studies allow great flexibility in investigating multiple exposures and multiple outcomes; the location, as Greece is a warm, sunny country with inhabitants exposed to high-O 3 concentrations; the large number of students and the relatively long period of follow up; the limited number of participants lost to follow-up or with incomplete data; the personal O 3 monitoring; and the wealth of data collected over a variety of respiratory morbidity indices using standardized procedures. Moreover, our results were robust after controlling for particles and under several sensitivity/subgroup (e.g., after considering only the asthmatic schoolchildren) analyses.

Conclusion
The study provides evidence that airway inflammation and the frequency of respiratory symptoms increase while lung function decreases with increased O 3 exposure in school children generally representative of the population of children 10-to 11-y-old in two large Southern European cities, where high-ozone concentrations are observed. Schoolchildren should be considered at a moderate risk of suffering respiratory health adverse effects following short-term exposure to the current levels of ambient O 3 , in line with what has been found in controlled human exposure studies at ambient concentrations. Focusing on asthmatic children may lead to an underestimation of the real burden of O 3 induced respiratory adverse events.