6.6-Hour Inhalation of Ozone Concentrations from 60 to

87 Parts per Billion in Healthy Humans

Edward S. Schelegle1, Christopher A. Morales1, William F. Walby1, Susan Marion2, and Roblee P. Allen3

1

Department of Anatomy, Physiology, and Cell Biology, University of California Davis School of Veterinary Medicine, Davis; 2California National

Primate Research Center, University of California Davis, Davis; and 3

Department of Internal Medicine, University of California Davis Medical Center,

Sacramento, California

Rationale: Identification of the minimal ozone (O3) concentration

and/or dose that induces measurable lung function decrements in

humans is considered in the risk assessment leading to establishing

an appropriate National Ambient Air Quality Standard for O3 that

protects public health.

Objectives: To identify and/or predict the minimal mean O3 concentration that produces a decrement in FEV1 and symptoms in healthy

individuals completing 6.6-hour exposure protocols.

Methods: Pulmonary function and subjective symptoms were measured in 31 healthy adults (18–25 yr, male and female, nonsmokers)

who completed five 6.6-hour chamber exposures: filtered air and

four variable hourly patterns with mean O3 concentrations of 60, 70,

80, and 87 parts per billion (ppb).

Measurements and Main Results: Compared with filtered air, statistically significant decrements in FEV1 and increases in total subjective

symptoms scores (P , 0.05) were measured after exposure to mean

concentrations of 70, 80, and 87 ppb O3. Themean percent change in

FEV1 (6standard error) at the end of each protocol was 0.80 6 0.90,

–2.72 6 1.48, 25.34 6 1.42, 27.02 6 1.60, and 211.42 6 2.20% for

exposure to filtered air and 60, 70, 80, and 87 ppb O3, respectively.

Conclusions: Inhalation of 70 ppb O3 for 6.6 hours, a concentration

below the current 8-hour National Ambient Air Quality Standard of

75 ppb, is sufficient to induce statistically significant decrements in

FEV1 in healthy young adults.

Keywords: ozone; clinical study; exposure assessment; human

Ozone is the primary oxidant found in photochemical air pollution and is one of the six criteria air pollutants identified in the

1971 United States Clean Air Act as adversely affecting public

health. Human clinical exposure studies have played an important role in the risk assessment required to set the National

Ambient Air Quality Standard (NAAQS) for ozone. These

clinical exposure studies have been designed to expose defined

subpopulations of individuals while varying ozone concentrations, exposure duration, and minute ventilations. A central

question inherent in these studies has been as follows: What is

the minimal mean ozone concentration that produces a statistically significant decrement in FEV1 and other markers of response?

Early clinical studies focused on manipulating ozone concentration and/or minute ventilation (V_ E) by adjusting exercise

workloads, while limiting the duration of exposure to 2.5 hours

or less (1–4). The minimal mean ozone concentration that

produced a statistically significant decrement in FEV1 in healthy

male subjects in these early studies was shown to be 120 ppb

using a 2.5-hour protocol and heavy intermittent exercise (15-

min periods of rest and exercise with exercise V_ E of 65 L/min)

(3) and 300 ppb using a 1-hour protocol and heavy continuous

exercise (exercise V_ E of 66 L/min) (4). Subsequently, Schelegle

and Adams (5) observed a statistically significant decrement in

FEV1 in endurance athletes who completed a 1-hour competitive simulation (mean V_ E of 86.6 L/min) while breathing

180 ppb ozone. More recently several investigators have extended exposure duration to 6.6 hours, using a protocol initially

described by Folinsbee and colleagues (6). This protocol contains

six 50-minute exercise periods with minute ventilation maintained at 8 L/min/L of FVC (V_ E of approximately 40 L/min). As

noted by Folinsbee and colleagues (6) and McDonnell and

colleagues (7), this level of exertion was ‘‘intended to simulate

work performed during a day of heavy to severe manual labor in

outdoor laborers.’’ Folinsbee and colleagues (6), Horstman and

colleagues (8), and Adams (9, 10) have used this 6.6-hour protocol

to examine ozone-induced responses in healthy young adults

exposed to ozone concentrations ranging from 40 to 120 ppb. Of

these, the studies conducted by Adams (9–11) have played an

important role in the 2007 exposure/risk assessment conducted by

the United States Environmental Protection Agency and the

establishment of a new NAAQS for ozone in 2008. Adams (9)

observed statistically significant FEV1 decrements and respiratory

symptoms at 80 ppb. Although lung function decrements and

respiratory symptoms in the 60 ppb ozone protocols were not

statistically significant, the magnitude of the pulmonary function

decrements at 60 ppb ozone at 40 L/minute were consistent

with the trend observed at higher levels and some subjects had

‘‘notable’’ FEV1 decrements (i.e., .10% FEV1) at 60 ppb.

The current study further examines the minimal mean ozone

concentration that produces a statistically significant decrement

in FEV1 and symptoms in healthy individuals completing five

6.6-hour exposure protocols with variable ozone concentration

AT A GLANCE COMMENTARY

Scientific Knowledge on the Subject

The acute inhalation of ambient concentrations of ozone

induces several health effects including airway irritation

and inflammation, decrements in pulmonary function, and

symptoms of respiratory discomfort.

What This Study Adds to the Field

This study identifies 70 ppb as the mean concentration of

ozone averaged over 6.6 hours that results in a statistically

significant decrement in FEV1 and presents an empirically

validated model that predicts the onset of pulmonary

responses induced by any combination of ozone concentration, minute ventilation, and exposure duration.

(Received in original form September 23, 2008; accepted in final form May 13, 2009)

Supported by an unrestricted gift from the American Petroleum Institute.

Correspondence and requests for reprints should be addressed to Edward S.

Schelegle, Ph.D., Department of Anatomy, Physiology, and Cell Biology, University

of California Davis, 1 Shields Avenue, Davis, CA 95616. E-mail: esschelegle@

ucdavis.edu

Am J Respir Crit Care Med Vol 180. pp 265–272, 2009

Originally Published in Press as DOI: 10.1164/rccm.200809-1484OC on May 15, 2009

Internet address: www.atsjournals.org
profiles. In addition, the variable ozone concentration profiles

were specifically designed to examine the relative importance of

mean ozone concentration over the entire exposure period

versus peak dose rate in determining peak pulmonary function

decrements and symptoms during and after exposure. This was

accomplished by adjusting the mean and peak ozone concentrations for each exposure protocol. The mean ozone concentrations of the five protocols were 0, 60, 70, 80, and 87 ppb. Each

of the four stepwise ozone concentration profiles (60, 70, 80, and

87 ppb ozone) had peak ozone concentrations in the fourth hour

of exposure with peaks of 90, 90, 150, and 120 ppb, respectively.

The dose of onset (Dos) of ozone-induced decrements in FEV1

was derived from the combined individual subject FEV1 and

cumulative dose data, using an iterative process involving leastsquares linear regression analysis (12). The derived value of

Dos that represents the cumulative dose of ozone at which

approximately half the subjects have developed pulmonary

responses and half have not was then combined with other

published (12) values of Dos to predict the onset of ozoneinduced pulmonary responses in the healthy young subjects at

multiple combinations of ozone concentration, exposure duration, and minute ventilations. The results of this study have

been previously reported in the form of an abstract (13).

METHODS

Subject Recruitment

Thirty-one young adults (16 females and 15 males), ages 18–25 years,

participated in the study. Subjects were solicited volunteers from the

University of California, Davis, or surrounding community. Each

subject was informed of study risks and subsequently signed a consent

form approved by the Institutional Human Subjects Review Board

(IRB) of the University of California, Davis. Individuals with a history

of cardiovascular disease or respiratory ailments (i.e., asthma, seasonal

allergies) were not allowed to participate in the study. Subjects were

nonsmokers and did not live in a high air pollution area 6 months

before participation in the study. All subjects were engaged in a regular

program of aerobic training to ensure their ability to complete the

exercise protocols. All subjects underwent preexperimental screening

to determine normal pulmonary function (Table 1) and filled out

a general health questionnaire, both of which were reviewed by the

project physician before being included in the study. In addition,

female subjects were asked to perform a urine pregnancy test to make

sure they were not pregnant at the time of enrollment.

Experimental Design

All exposures were performed in a free-standing 9 3 10 3 8 ft stainless

steel environmental chamber (model 1328-M; Vista Scientific, Ivyland,

PA). The chamber and its operation have been previously described by

Adams (9). The five 6.6-hour chamber exposures completed by each

subject were composed of six 50-minute exercise bouts at a mean

equivalent ventilation rate of 20 L/minute/m2 body surface area (BSA)

(9). The 50-minute exercise bouts were done alternately on a cycle

ergometer and treadmill. A 35-minute lunch break took place after

completion of Hour 3 and was taken at rest in the chamber at the Hour

3 O3 concentration. Temperature and relative humidity were maintained between 21 and 258C and between 40 and 60%, respectively.

The exposure regimens were composed of filtered air and four varying

hourly O3 concentrations averaging 60, 70, 80, and 87 ppb. The

stepwise patterns of O3 concentration are given in Table 2. Ozone

concentration was monitored continuously with a Dasibi monitor

(model 1003H; Dasibi Instruments Inc, Glendale, CA) calibrated

according to the ultraviolet absorption photometric method, traceable

to a National Institute of Standards and Technology standard photometer, at the Primate Research Center of the University of California,

Davis. The protocols were conducted in single-blind fashion and

completed by each subject in random order, with a minimum of 7 days

intervening between protocols.

Subjects performed two to four forced expiratory maneuvers

immediately before and after each experimental exposure, during the

last 10 minutes of each hour and at 1 and 4 hours postexposure, using

a portable computer-based spirometer (SpiroVision-3; FUTUREMED

Inc., Granada Hills, CA). The FVC and FEV1 values were selected on

the basis of American Thoracic Society guidelines (14).

Minute ventilation (V_ E), tidal volume (VT), breathing frequency (f),

expired gas temperature, heart rate, and subjective symptoms were

monitored as previously described (9). Subjects were asked to rate the

severity of each of four symptoms: throat tickle, cough, shortness

of breath, and pain on deep inspiration. Each symptom was rated

according to a severity scale (ranging from 0, not present, to 40, severe)

as previously described (15). Total subjective symptoms (TSS) score

was calculated as the sum of the severity ratings for the four individual

symptoms.

Statistical Procedures

The effect of exposure on FVC, FEV1, and FEV1/FVC was expressed

as the percent change from the preexposure value. Similarly, f, VT, and

V_ E are presented as percent change from the initial value obtained at

7–10 minutes of the first exercise period. TSS was analyzed as absolute

changes from zero. All data are expressed as means (standard error).

The effects of gas concentration and exposure time were determined by

mixed model two-way analysis of variance (ANOVA) with repeated

measures (P , 0.05) (SAS software; SAS Institute, Cary, NC), using

TABLE 1. FEMALE AND MALE SUBJECT PHYSICAL

CHARACTERISTICS AND BASELINE PULMONARY

FUNCTION MEASURES

Characteristic Females (n 5 16) Males (n 5 15)

Age, yr 21.4 (0.6) 21.4 (0.5)

Height, m 1.68 (0.02) 1.82 (0.02)

Weight, kg 65.1 (2.8) 81.0 (2.8)

BSA, m2 1.73 (0.05) 2.04 (0.04)

FVC, L 4.16 (0.19) 5.72 (0.19)

FEV1, L 3.43 (0.13) 4.67 (0.18)

FEV1/FVC, % 82.8 (1.9) 81.7 (1.5)

FEF25–75, L/s 3.35 (0.15) 4.48 (0.29)

PEF, L/s 7.04 (0.17) 9.96 (0.46)

Definition of abbreviations: BSA 5 body surface area; FEF25–75 5 forced

expiratory flow between 25 and 75% FVC; PEF 5 peak expiratory flow.

Values represent means (SE).

TABLE 2. TARGET AND ACTUAL MEAN OZONE

CONCENTRATIONS EXPRESSED AS PARTS PER BILLION FOR

THE FIVE 6.6-HOUR EXPOSURE PROTOCOLS

Exercise Period

Protocol

Target/

Actual 1 2 3 4 5 6 Mean

FA Target 0 0 0 0 0 0 0

Actual 1 (0) 1 (0) 1 (0) 1 (0) 1 (0) 1 (0) 1 (0)

60 ppb† Target 40 70 70 90 50 40 60

Actual 43 (1) 72 (1) 73 (1) 91 (1) 54 (1) 42 (1) 63 (1)

70 ppb‡ Target 50 70 80 90 80 50 70

Actual 52 (1) 73 (1) 82 (1) 92 (0) 81 (1) 54 (1) 72 (1)

80 ppbx Target 30 70 100 150 80 50 80

Actual 33 (1) 71 (1) 101 (1) 147 (1) 84 (1) 52 (1) 81 (1)

87 ppbk Target 40 80 90 120 100 90 87

Actual 42 (1) 81 (1) 93 (1) 119 (1) 102 (1) 91 (1) 88 (1)

Definition of abbreviation: FA 5 filtered air.

Actual values represent means (SE); n 5 31. † Pattern is modified from Adams (9), with the ozone concentration in the third

and fourth exercise period switched. ‡ Daily pattern observed in Little Rock, Arkansas metropolitan area (A. Lefohn,

personal communication). x Pattern is the Adams (9) stepwise 80 ppb protocol and provides a means of

direct comparison with previous published findings.

k Daily pattern observed in San Bernardino, California metropolitan area

(A. Lefohn, personal communication).

266 AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE VOL 180 2009
the procedures described by Littell and colleagues (16). There are two

steps in performing a mixed model two-way ANOVA with repeated

measures (16). In the first step we determined the best fit of the data to

one of several within-subject covariance structures using the Akaike

information criterion (AIC) and the Schwarz Bayesian criterion (SBC).

After fitting the data to unstructured, Toeplitz, compound symmetry,

heterogeneous compound symmetry, heterogeneous first-order autoregressive, first-order autoregressive, and heterogeneous covariance

structures both the AIC and SBC indicated that the Toeplitz covariance structure provided the best fit to the data. In the second step

we analyzed the time and protocol effects by estimating and comparing

means, initially using least-squares means and then using a Tukey

adjustment (SAS Institute).

Having analyzed the data in a manner that allowed for the evaluation of the time and mean concentration effects we reanalyzed the

data, focusing on the effect that each protocol had on the immediate

postexposure FEV1 compared with the filtered air protocol. To make

this comparison we used both parametric and nonparametric tests.

The parametric analysis followed the same procedures as those used

for the whole data set described previously, except that a Dunnett’s

adjustment was used to compare means. The Dunnett’s test limits the

number of comparisons to those between the control group (in this case

the filtered air protocol) and each experimental protocol. The nonparametric approach used the Friedman test, which is the nonparametric equivalent of a repeated measures ANOVA on ranks, with post

hoc comparisons of mean ranks using Dunnett’s test (17).

To further examine the time course of the FEV1 response we

identified the dose of onset (Dos), using a regression approach similar

to the one we previously described and applied to breathing pattern

data (12). In brief, we plotted the percent change in FEV1, corrected

for filtered air (FA) responses, for all the subjects combined against

the cumulative dose of O3 (micrograms) for each exposure protocol.

Dos was determined by an iterative process involving least-squares

linear regression (Microsoft Excel X; Microsoft Corporation, Redmond, WA). In each iteration step two lines were fit to the percent

change in FEV1 and cumulative dose. In the first iteration the first

region (region 1) of the data that was fit included the first 31 data

points. The second region (region 2) that was fit began at the 32nd

point and included all the cumulative dose points greater than this to

the end of the protocol. In the next iteration a point was added to

region 1 and subtracted from region 2. This iterative process was

continued until region 2 consisted of the data from the last 31 points.

With each iteration step the slope, intercept, and correlation coefficient were calculated for each region. In addition, the difference in

slope of regions 1 and 2, and the average correlation coefficient of

regions 1 and 2, were calculated. The point at which the maximum in

the correlation coefficient of region 1, the average correlation coefficient of regions 1 and 2, and the difference in slopes of region 1

and 2 occurred was determined and averaged to obtain the estimated

Dos.

RESULTS

A summary of the male and female subjects’ characterization

data is given in Table 1. All the subjects were engaged in some

form of regular aerobic activity. One male subject was a competitive cyclist at the time of the study. All the subjects were

healthy and had normal pulmonary functions, with the baseline

value of FEV1/FVC% ranging from 69.8 to 96.2%.

The group mean exercise V_ E and estimated overall mean

V_ E (includes estimated resting V_ E [18]) values for the five 6.6-

hour protocols are given in Table 3. Resting V_ E was estimated

using regression equations derived from the data of Aitken

and colleagues (18) that relate resting V_ E to body surface area

for college-age males [resting V_ E 5 7.61(BSA)] and females

[resting V_ E 5 8.05(BSA)]. There were no statistically significant differences in exercise V_ E with regard to time of

exposure or protocol. In addition, there was no statistically

significant difference in the estimated overall mean V_ E for the

five protocols.

The group mean ozone concentrations during each exercise

period, as well as the average ozone concentration for each

protocol, are given in Table 2, whereas the mean cumulative

inhaled dose (CD, mg) of ozone is plotted against time of

exposure in Figure 1. The ozone concentrations (ppb) during

each exercise period for all four ozone protocols were significantly greater than the background levels measured in the FA

protocol. Comparing across protocols, whenever the target

ozone concentration was set to be different compared with

any other protocol the measured values for this comparison

were found to be significantly different. The inhaled dose rate

(DR, mg/min) during each exercise period for all four ozone

protocols was significantly greater than the background levels

measured in the FA protocol. In contrast to ozone concentration the DR during each exercise period was significantly

different across ozone protocols only when the difference in

the target ozone concentration was greater than or equal to

20 ppb. All possible comparisons of CD at the end of each protocol were significant different. However, the time during exposure at which the CD became significantly different between

ozone protocols varied (Figure 1). The CD for the 60 ppb exposure protocol became significantly different from the 70, 80,

and 87 ppb ozone exposure protocols during the fifth, fourth,

and third exercise periods, respectively. The CD for the 70 ppb

exposure protocol became significantly different from the 80

and 87 ppb ozone exposure protocols during the fourth exercise

period. The CD for the 80 ppb exposure protocol became

significantly different from the 87 ppb ozone exposure protocol

during the fifth exercise period.

TABLE 3. GROUP MEAN EXERCISE MINUTE VENTILATION AND

ESTIMATED OVERALL MINUTE VENTILATION* FOR THE FIVE

EXPOSURE PROTOCOLS

V_ E

Protocol Exercise Estimated Overall

FA 39.3 (0.9) 33.4 (0.8)

60 ppb 38.5 (0.9) 32.8 (0.7)

70 ppb 38.6 (1.0) 32.8 (0.8)

80 ppb 38.9 (1.0) 33.1 (0.8)

87 ppb 38.4 (0.9) 32.7 (0.7)

Definition of abbreviation: V_ E 5 minute ventilation.

Values represent means (SE).

* Inclusive of rest and lunch periods.

Figure 1. Diagram of mean group values for cumulative dose of ozone

(micrograms) against time of exposure for each of the five protocols.

Values represent means 6 SEM.

Schelegle, Morales, Walby, et al.: 6.6-Hour Inhalation of Ozone 267
Pulmonary Function and Symptom Responses

The mean responses for the percent change in FVC, percent

change in FEV1, percent change in FEV1/FVC%, and TSS

are shown in Figure 2. In comparison with the FA protocol

statistically significant decrements in the percent change in FVC

were present after the fourth and sixth exercise periods of the

80 ppb protocol and after the fifth and sixth exercise periods and

1 hour postexposure of the 87 ppb protocol (Figure 2A). In

comparison with the FA protocol the 70, 80, and 87 ppb

exposure protocols resulted in statistically significant decrements in the percent change in FEV1 (Figure 2B). These statistically significant differences occurred after the sixth exercise

period in the 70 ppb protocol; after the fourth, fifth, and sixth

exercise periods and 1 hour postexposure of the 80 ppb

protocol; and after the fifth and sixth exercise periods and

1 hour postexposure of the 87 ppb protocol. In comparison with

the FA protocol, statistically significant decrements in the

percent change in FEV1/FVC% were present only after the

fifth and sixth exercise periods of the 87 ppb protocol (Figure

2C). In comparison with the FA protocol the 70, 80, and 87 ppb

exposure protocols resulted in statistically significant increases

in TSS (Figure 2D). In each of these three exposure protocols

the statistically significant increases in TSS occurred after the

fifth and sixth exercise periods. In all the protocols pulmonary

function and symptoms returned to preexposure levels within

4 hours of the end of exposure.

In comparison with the FA protocol the inhalation of ozone

during the 60 ppb protocol did not result in a statistically

significant decrement in percent change in FVC, percent change

in FEV1, percent change in FEV1/FVC, or TSS at any time

point. Examination of the percent change in FEV1 shows that

the maximal difference between the FA and 60 ppb protocol

occurred after the sixth exercise period (Figure 2B). The

magnitude of this difference was 3.52 6 1.52% (mean 6 SE)

and was the result of 11 subjects who had FEV1 decrements

greater than 5% compared with the FA protocol (11.42 6

2.62%). To increase the power of our analysis we limited the

number of mean comparisons and narrowed the hypothesis

being tested by restricting our analysis to immediate postexposure FEV1 data and only comparing each ozone protocol

with the filtered air protocol. In addition, we used both parametric and nonparametric tests in this restricted analysis. The

distribution of the percent change in FEV1 at 6.6 hours for each

protocol is illustrated using histograms in Figure 3. While

increasing the power of the analysis both parametric and

nonparametric tests provided the same result as the more global

two-way ANOVA with repeated measures (Table 4).

Dose at Onset

We were able to obtain reliable estimates of Dos, using the

pooled FEV1 from the 80 and 87 ppb ozone exposure protocols

and when all of the FEV1 data was combined, but not from the

Figure 2. Mean responses of 31 healthy young adult subjects to various time–concentration profiles during, and 1 and 4 hours after, 6.6-hour

ozone exposure. (A) FVC; (B) FEV1; (C) FEV1/FVC, all percent change from baseline; and (D) total subjective symptoms scores. Asterisks indicate

a statistically significant difference from filtered air (FA) at the same time point. Note: Only a portion of the subjects completed the 4-hour

postexposure measurements: FA (n 5 17); 60 ppb (n 5 15); 70 ppb (n 5 15); 80 ppb (n 5 15); and 87 ppb (n 5 13). Values represent means 6

standard error of the mean.

268 AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE VOL 180 2009
pooled FEV1 data from the 60 and 70 ppb ozone exposure

protocols. The inability to estimate Dos using the FEV1 data

from the 60 and 70 ppb ozone exposure protocols is most likely

because less than one third of the subjects had changes in FEV1

greater than 5% in either of these protocols. The estimated

values of Dos from the 80 and 87 ppb ozone exposure protocols

are 1,374 and 1,326 mg of ozone, respectively. The estimated

value of Dos from all of the FEV1 data combined was 1,362 mg

of ozone.

DISCUSSION

The U.S. Clean Air Act defines a primary air quality standard to

protect the public health, while allowing for an adequate margin

of safety. An Ozone NAAQS was first established on April 30,

1971 and subsequently reviewed and revised in 1979, 1997, and

2008. The NAAQS for ozone established in 1971 and 1979 were

based on a peak 1-hour average. Subsequently, in 1997, in

an effort to address the broad multiple-hour elevations seen

in ambient ozone concentration in some urban and suburban

environments, the NAAQS for ozone was revised to an 8-hour

average concentration of 0.08 ppm for the fourth highest

average over 3 years. More recently the NAAQS for ozone

was revised to an 8-hour average concentration of 0.075 ppm for

the fourth highest average over 3 years. In the current study the

mean 6.6-hour ozone concentration of the four ozone protocols

brackets the current NAAQS. We observed statistically significant decrements in FEV1 and TSS associated with the 70, 80,

and 87 ppb protocols, but not the 60 ppb protocol. In addition,

there were statistically significant decrements in FVC in the 80

and 87 ppb protocols and in FEV1/FVC in the 87 ppb protocol.

These findings lower the mean ozone concentration at which

statistically significant decrements in FEV1 have been observed

during a 6.6-hour exposure protocol to 70 ppb.

Figure 3. Histograms of percent change in FEV1 after 6.6-hour exposure to (A) filtered air, (B) 60 ppb O3, (C) 70 ppb O3, (D) 80 ppb O3, and (E)

87 ppb O3 for both female subjects (solid column portions) and male subjects (shaded column portions). Values represent means (SEM).

TABLE 4. PROBABILITY VALUES OBTAINED BY THREE

STATISTICAL APPROACHES TO ANALYZE FEV1 RESPONSE

AFTER FOUR 6.6-HOUR EXPOSURE PROTOCOLS COMPARED

WITH FILTERED AIR

Statistical Test

Comparison

Two-way ANOVA

with Tukey-Kramer

One-way ANOVA

with Dunnett’s

Test

Friedman’s

Test with Dunnett’s

Test

FA vs. 60 ppb 0.8 0.2 .0.05

FA vs. 70 ppb 0.0016 0.0023 ,0.01

FA vs. 80 ppb ,0.0001 0.0002 ,0.001

FA vs. 87 ppb ,0.0001 ,0.0001 ,0.0001

Definition of abbreviation: ANOVA 5 analysis of variance.

Schelegle, Morales, Walby, et al.: 6.6-Hour Inhalation of Ozone 269
These findings are consistent with the results of Adams (9),

who reported statistically significant decrements in FEV1 using

a 6.6-hour protocol with a mean ozone concentration of 80 ppb,

but not 40 or 60 ppb. Adams (9) obtained the same result while

using both constant ozone concentrations of either 60 or 80 ppb

over the exposure period, and variable stepwise ozone concentration profiles similar but not identical to those used in the

current study. There has been some concern expressed in the

literature (19) that the univariate two-way ANOVA with

repeated measures followed by Scheffe ́’s post hoc test used by

Adams in 2006 (9) indicating no statistically significant effect on

FEV1 in the 60 ppb protocols lacked sufficient statistical power

to guarantee that this finding did not represent a false negative

(type II error). It has been suggested that limiting the analysis

to the immediate postexposure FEV1 data and restricting the

mean comparisons to the filtered air control would increase the

power of the analysis and allow for the detection of significant

differences when differences between means are small (18).

We recognize the validity of this suggestion and agree that

limiting the scope of the hypothesis being tested can increase

the power of the statistical approach; however, we also recognize that when doing so caution should be exercised to ensure

that the approach used is consistent with the original study

design. In this case the individual subject FEV1 responses

induced by each exposure protocol are not independent and

therefore any analysis should consider the effect of multiple

comparisons. To address these concerns in the current paper we

analyzed our data using a mixed-model two-way ANOVA with

repeated measures followed by Tukey’s post hoc test and then

analyzed the immediate postexposure FEV1 data using both

parametric and nonparametric approaches. The mixed-model

two-way ANOVA with repeated measures followed by Tukey’s

post hoc test provides the ability to directly address the covariance structure of the data and greatly enhances the ability to

analyze repeated measures data by providing valid standard

errors and efficient and powerful comparisons of means within

a global analysis (16). This greatly improves the ability to

examine time effects within protocols and protocol effects at

multiple time points. Our analysis of the immediate postexposure FEV1 data using both parametric and nonparametric

statistics, while correcting for the inherent multiple comparisons

in the original study design, optimized the power of the analysis

by limiting the mean comparisons to those between the filtered

air protocol and the 60, 70, 80, and 87 ppb exposure protocols.

Furthermore, we also used a nonparametric statistic that is

appropriate if the within-subject variance is not normally

distributed (17). We obtain a similar result regardless of our

statistical method, with the 60 ppb exposure protocol not being

significantly different from filtered air, whereas the 70, 80, and

87 ppb exposure protocols were significantly different from

filtered air. Although recognizing the consistency of our statistical analyses we point out, as did Adams (11), that there is

a subset of responsive subjects that did respond to the 60 ppb

protocol in excess of a 10% decrement in FEV1. In addition, it is

important to note that the previous study conducted by Adams

(11) and the current study use a 6.6-hour protocol, which is

1.4 hours less than the 8-hour NAAQS and that if the 60 ppb

ozone protocols were extended greater decrements might be

achieved. A counterpoint to this possibility is the fact the mean

overall ventilations in Adams (9) and this study are equal to or

greater than mean ventilations that might be encountered

during a day of heavy to severe manual labor among the construction workers observed by Linn and colleagues (20) and

that this represents the higher end of ventilations that might

be encountered in the normal population for this prolonged

period.

In the current study, the variable stepwise profile of ozone

concentration differed from protocol to protocol in such a way

that the peak ozone concentration did not correlate with the

mean ozone concentration over the entire protocol (Table 2).

The clearest example of this and the one with demonstrated

consequences are the 80 and 87 ppb protocols in which the peak

1 hour (4.6 to 5.6 h) values were 150 and 120 ppb ozone,

respectively. The 80 ppb protocol also started and ended at

a lower ozone concentration (30 and 50 ppb ozone) than the

87 ppb protocol (40 and 90 ppb ozone). The net result was that

the cumulative dose for these two protocols did not become

significantly different from each other until the final hour of

exposure (see Figure 1), with the dose rate becoming significantly greater in the 80 ppb protocol during the fourth exercise

period and then becoming significantly less during the fifth and

sixth exercise periods. This pattern of exposure resulted in

decrements in FEV1 becoming statistically significant 1 hour

earlier (4.6 vs. 5.6 h) in the 80 ppb protocol compared with the

87 ppb protocol (Figure 2B). This pattern then resulted in

a plateau in FEV1 decrements in the 80 ppb protocol, whereas

FEV1 decrements continued to increase in the 87 ppb protocol

(Figure 2B). These observations, in combination with a delay in

onset of response of approximately 3 hours (Figure 2) in the face

of an increasing cumulative dose, suggest that there exists a

complex interaction between time and dose rate at the level of

the individual subject and cohort studied.

Several studies support the hypothesis that ozone-induced

rapid shallow breathing and decrements in inspiratory capacity

and FEV1 are mediated by lung C-fibers (21–23) and could be

expected to have similar time courses. Using breathing pattern

data collected from 97 healthy male and female subjects during

ozone exposure protocols of shorter duration, higher ozone

concentrations, and continuous exercise of greater intensity

than those used in the current study, we identified a distinct

delay and response phase in the development of ozone-induced

tachypnea (12). We found that the delay phase was dependent

on reaching a dose of onset (Dos) and that the value of Dos was

not influenced by ozone concentration or duration of exposure

and only mildly influenced by changing V_ E. The consequence of

this relationship is that if V_ E is held constant the higher the mean

ozone concentration the shorter the time to reach the threshold

for the onset of response. In addition, we observed that the magnitude of tachypnea that developed after Dos was reached correlated with dose rate and not the cumulative or effective inhaled

dose. Also of considerable importance was the observation that

the magnitude of Dos was not correlated with the magnitude of

tachypnea. We proposed that the development of decrements in

FVC and FEV1 may follow a similar time course and cited

previous 6.6-hour exposure protocols to support this possibility.

The plot of the group mean decrement in FEV1 versus cumulative inhaled dose (Figure 4) supports the notion that a Dos is

clearly present in the FEV1 data in the current study. In addition, the plateau of FVC and FEV1 decrements in the 80 ppb

protocol, despite the fact that cumulative inhaled dose continues to increase, supports the notion that after Dos is reached the

magnitude of response is a function of dose rate.

Using a similar regression analysis approach for deriving Dos

from breathing frequency data, we determined Dos on the basis

of the combined individual FEV1 data. The derived Dos, using

all the pooled FEV1 data, was 1,362 mg of ozone. This value of

Dos is greater than the values of Dos derived in our previous

analysis (12). This difference may be related to numerous

factors, for example, the plot of Dos from this and our previous

analysis against V_ E further suggests that Dos is a function of

V_ E (Figure 5A). Dos is not only useful in providing a better

understanding of the kinetics of ozone-induced pulmonary

270 AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE VOL 180 2009
responses but provides insights into a component that contributes to the individual or group responsiveness to ozone. Given

the relationship between Dos and V_ E (Figure 5A) it is possible

to predict the average maximal dose of ozone at which there

is no pulmonary function decrement for exposures varying

greatly in ozone concentration, duration of exposure, and

minute ventilation (Figure 5B). The three-dimensional surface

defined by this relationship (Figure 5B) provides a tool for

predicting the maximal ozone exposure that approximately half

of healthy individuals could experience without demonstrating

functional responses. However, this relationship needs to be

further validated, especially with studies using lower minute

ventilations in combination with ambient ozone concentrations.

It is also of critical importance to gain a better understanding of

how the airway environment changes at the onset of decrements

in lung function and subjective symptoms.

Conflict of Interest Statement: None of the authors has a financial relationship

with a commercial entity that has an interest in the subject of this manuscript

Acknowledgment: The authors thank Allen S. Lefohn, Ph.D. (A.S.L. & Associates,

Helena, MT) and Milan Hazucha, M.D., Ph.D. (Center for Environmental

Medicine, Asthma, and Lung Biology, Chapel Hill, NC) for assistance in designing

the exposure protocols. The authors thank Emily Wong, Wyatt Hesemeyer,

Denise Veloria, Conrad Sherby, Carlie Allison, Courtney Gertler, Tyler Dibble,

Harpul Bhamra, and Cemal Ozemek for assistance in running experimental

protocols and in data analysis.

References

1. Silverman F, Folinsbee LJ, Barnard J, Shephard RJ. Pulmonary function

changes in ozone-interaction of concentration and ventilation. J Appl

Physiol 1976;41:859–864.

2. Folinsbee LJ, Drinkwater BL, Bedi JF, Horvath SM. The influence of

exercise on the pulmonary function changes due to low concentrations of ozone. In: Folinsbee LJ, editor. Environmental stress. New

York: Academic Press; 1978. pp. 125–145.

3. McDonnell WF, Hortsman DH, Hazucha MJ, Seal E Jr, Haak ED,

Salaam SA, House DE. Pulmonary effects of ozone exposure during

exercise: dose–response characteristics. J Appl Physiol 1983;54:1345–

1352.

4. Adams WC, Savin WM, Christo AE. Detection of ozone toxicity during

continuous exercise via the effective dose concept. J Appl Physiol 1981;

51:415–422.

5. Schelegle ES, Adams WC. Reduced exercise time in competitive

simulations consequent to low level ozone exposure. Med Sci Sports

Exerc 1986;18:408–414.

6. Folinsbee LJ, McDonnell WF, Horstman DH. Pulmonary function and

symptom responses after 6.6-hour exposure to 0.12 ppm ozone with

moderate exercise. JAPCA 1988;38:28–35.

7. McDonnell WF, Kehrl HR, Abdul-Salaam S, Ives PJ, Folinsbee LJ,

Devlin RB, O’Neil JJ, Horstman DH. Respiratory response of

humans exposed to low levels of ozone for 6.6 hours. Arch Environ

Health 1991;46:145–150.

8. Horstman DH, Folinsbee LJ, Ives PJ, Abdul-Salaam S, McDonnell WF.

Ozone concentration and pulmonary response relationships for 6.6-

hour exposures with five hours of moderate exercise to 0.08, 0.10, and

0.12 ppm. Am Rev Respir Dis 1990;142:1158–1163.

9. Adams WC. Comparison of chamber 6.6-h exposures to 0.04–0.08 ppm

ozone via square-wave and triangular profiles on pulmonary responses.

Inhal Toxicol 2006;18:127–136.

10. Adams WC. Comparison of chamber and face mask 6.6-hour exposure

to 0.08 ppm ozone via square-wave and triangular profiles on pulmonary responses. Inhal Toxicol 2003;15:265–281.

11. Adams WC. Comparison of chamber and face-mask 6.6-hour exposures

to ozone on pulmonary function and symptoms responses. Inhal Toxicol

2002;14:745–764.

12. Schelegle ES, Walby WF, Adams WC. Time course of ozone-induced

changes in breathing pattern in healthy exercising humans. J Appl

Physiol 2007;102:688–697.

13. Schelegle ES, Morales CA, Walby WF, Allen RP. 6.6 Hour human

ozone exposures with varying time–concentration profiles above and

below the current air quality standard [abstract]. Am J Respir Crit

Care Med 2008;177:A428.

14. Miller MR, Hankinson J, Brusasco V, Burgos F, Casaburi R, Coates A,

Crapo R, Enright P, van der Grinten CP, Gustafsson P, et al.

Standardisation of spirometry. Eur Respir J 2005;26:319–338.

15. Adams WC, Brookes KA, Schelegle ES. Effects of NO2 alone and in

combination with O3 on young men and women. J Appl Physiol 1987;

62:1698–1704.

Figure 4. Scatter plot of group mean decrements in FEV1 against the

total cumulative dose of ozone (micrograms). Note that there is an

inflection in the data between 1,300 and 1,400 mg of ozone. Values

represent means 6 SEM.

Figure 5. (A) Plot of dose of onset (Dos) derived previously by

Schelegle and colleagues (12) (solid squares) and in the current study

(solid circle) plotted against mean minute ventilation. (B) The threedimensional surface defined by the relationship between Dos and

minute ventilation that predicts the average cumulative dose of ozone

in an average young healthy adult, below which no appreciable FEV1

decrement occurs for exposures varying greatly in ozone concentration, duration of exposure, and minute ventilation. Dos is the cumulative dose of ozone at which approximately half the subjects have

developed pulmonary responses and half have not.

Schelegle, Morales, Walby, et al.: 6.6-Hour Inhalation of Ozone 271
16. Littell RC, Henry PR, Ammerman CB. Statistical analysis of repeated

measures data using SAS procedures. J Anim Sci 1998;76:1216–

1231.

17. Glantz SA. Primer of biostatistics, 5th ed. New York: McGraw-Hill;

2002. pp. 298–381.

18. Aitken ML, Franklin JL, Pierson DJ, Schoene RB. Influence of

body size and gender on control of ventilation. J Appl Physiol 1986;

60:1894–1899.

19. Brown JS, Bateson TF, McDonnell WF. Effects of exposure to 0.06 ppm

ozone on FEV1 in humans: a secondary analysis of existing data.

Environ Health Perspect 2008;116:1023–1026.

20. Linn WS, Spier CE, Hackney JD. Activity patterns in ozone-exposed

construction workers. J Occup Med Toxicol 1993;2:1–14.

21. Hazucha MJ, Bates BV, Bromberg PA. Mechanism of action of ozone

on the human lung. J Appl Physiol 1989;67:1535–1541.

22. Schelegle ES, Elderidge MW, Cross CE, Walby WF, Adams WC.

Differential effects of airway anesthesia on ozone-induced pulmonary

responses in human subjects. Am J Respir Crit Care Med 2001;163:

1121–1127.

23. Passannante AN, Hazucha MJ, Bromberg PA, Seal E, Folinsbee L,

Koch G. Nociceptive mechanisms modulate ozone-induced human

lung function decrements. J Appl Physiol 1998;85:1863–1870.

272 AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE VOL 180 2009