An acute exposure to ozone impairs human olfactory functioning

Author’s Accepted Manuscript An acute exposure to ozone impairs human olfactory functioning Axel Muttray, Jan Gosepath, Florian Schmall, Jürgen Brieger, Otfried Mayer-Popken, Michael Melia, Stephan Letzel www.elsevier.com/locate/envres

PII: DOI: Reference:

S0013-9351(18)30368-2 https://doi.org/10.1016/j.envres.2018.07.006 YENRS7990

To appear in: Environmental Research Received date: 13 December 2017 Revised date: 11 June 2018 Accepted date: 3 July 2018 Cite this article as: Axel Muttray, Jan Gosepath, Florian Schmall, Jürgen Brieger, Otfried Mayer-Popken, Michael Melia and Stephan Letzel, An acute exposure to ozone impairs human olfactory functioning, Environmental Research, https://doi.org/10.1016/j.envres.2018.07.006 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

An acute exposure to ozone impairs human olfactory functioning

Axel Muttraya*, Jan Gosepathb, Florian Schmalla,b, Jürgen Briegerb, Otfried Mayer-Popkena, Michael Meliaa, Stephan Letzela

a

Institute of Occupational, Social and Environmental Medicine and University Medical Center of the Johannes

Gutenberg University Mainz Langenbeckstraße 1 D - 55131 Mainz Germany b

Department of Otolaryngology of the University Medical Center of the Johannes Gutenberg University Mainz

Langenbeckstraße 1 D - 55131 Mainz Germany

*

Corresponding author. Prof. Dr. Axel Muttray, Institute of Occupational, Social and Environmental Medicine,

University Medical Center of the Johannes Gutenberg University Mainz, Langenbeckstraße 1, D - 55131 Mainz, Germany. Tel.: 0049-6131-179148; fax: 0049-6131-179045. [email protected]

Abstract Introduction Ozone is a ubiquitous and irritant gas. We questioned whether an acute exposure to 0.2 ppm ozone impaired olfactory functioning. Methods Healthy, normosmic subjects were exposed according to a parallel group design either to 0.2 ppm ozone (n=15) or to sham (n=13) in an exposure chamber for two hours. Possible irritating effects were assessed by questionnaire (range 0-5). The detection threshold of n-butanol was measured with the Sniffin’ Sticks test before and after exposure. Olfactory thresholds were logarithmized and a two-way analysis of variance (ANOVA) with repeated measurements was carried out to test the effects of exposure (ozone vs. sham) and time (before vs. after exposure). Additionally, nasal secretions were taken at a preliminary examination and after exposure to determine interleukins 1ß and 8. Results No irritating effects to the upper airways were observed. In the ozone group, the median score for cough increased from 0 to 2 at the end of exposure (sham group 0 and 0, respectively, p<0.001). The ANOVA showed

a main effect for ozone exposure (F (1, 26) = 27.6, p= 0.0002), indicating higher olfactory thresholds in the ozone group. Concentrations of interleukins in nasal secretions did not increase following ozone exposure. Conclusions This study shows a clear impairment of olfactory functioning following an acute exposure to 0.2 ppm ozone.

Keywords: Ozone, exposure, human, nasal mucosa, olfactory threshold

1. Introduction The irritant gas ozone is the most reactive gas species to which humans are environmentally exposed to (Pryor, 1992). It is one of the major air pollutants known for its negative effects on different health issues (e.g. (Devlin et al., 2012; Ross et al., 2012; Turner et al., 2016)). In 1955, ground-level ozone concentration peaked at 0.68 ppm in downtown Los Angeles (South Coast Air Quality Management District, 1997). Outdoor ozone concentrations have considerably declined since the 1950s, but peak values still exceeded 0.2 ppm in urban and industrial areas in different countries (Achcar et al., 2013; Banta et al., 2011; Cercelaru et al., 2016; Chelani, 2013; Li et al., 2013; Paoletti et al., 2014). Peak concentrations in airplane cabins can exceed 0.2 ppm ozone (Bekö et al., 2015). For comparison, the US EPA has revised the primary and secondary ozone standard levels to 0.07 ppm (Environmental Protection Agency, 2015). The European Commission has fixed a target value at 120 µg/m³ (i.e. 0.06 ppm, maximum daily 8 hour mean, 25 days averaged over 3 years (European Commission, 2016)). The information threshold has been set an hourly average of 180 µg/m³. Human exposure by inhalation to ozone may increase with respiratory minute volume due to outdoor work or sports as a result of physical workload. An indoor exposure to ozone is possible at various workplaces. Ozone is produced in arc welding by the action of ultraviolet light on oxygen (Dennis et al., 2002). Ozone concentrations in the welder’s breathing zone depend on the type of welding and range up to 0.4 ppm (Fachausschuss Metallund Oberflächenbehandlung, 2009). Furthermore, ozone has been used in food processing, as an air, water, and waste disinfectant, in the laundry industry, in pulp mills, and as a means to improve air quality (Cardis et al., 2007; Hoffman et al., 2004; Martínez et al., 2011; Mokoena et al., 2011; Omer and Walker, 2011; Parga et al., 2003; Rice, 2012; Shaughnessy et al., 1994). Various applications in the medical field have been reported, too (Mokoena et al., 2011; Vrablik and Green-McKenzie, 2016). Laser printers and photocopiers with outdated technology emit relevant amounts of ozone (Evers and Nowak, 2006; Tuomi et al., 2000).

2

Animal studies and modeling suggest that the greatest ozone dose to the respiratory tract tissue of humans occurs in the terminal bronchioles (Medinsky and Bond, 2001). The irritant effects of ozone on the lower airways have been extensively investigated (e.g. (Environmental Protection Agency, 2013)). When breathing through the nose, approximately half of the inhaled ozone is removed from the inhaled air during contact with the surface area of the nasal cavity (Medinsky and Bond, 2001; Nikasinovic et al., 2003). After experimental exposure, ozone was found in nasal lavage fluid of human subjects (Hatch et al., 1994). The extracellular lining fluid (ELF) is a complex mixture of antioxidants, proteins and lipids (Rees et al., 2008) that serves as the first target and barrier for inhaled ozone. If a dose is high enough, ozone depletes the antioxidant level in the nasal ELF (Environmental Protection Agency, 2013). Then, ozone can quantitatively react with targets such as lipids and proteins. Secondary oxidation products include free radicals, peroxides and aldehydes. These substances can induce cell injury in the respiratory mucosa and inflammatory signaling (Prueitt and Goodman, 2016). Inflammatory effects on the human and primate nasal respiratory epithelium have been described in previous research (e.g. (Carey et al., 2011; Graham and Koren, 1990; Harkema et al., 1987)). In contrast, reports on possible toxic effects of the mammalians’ olfactory epithelia are rare. A two-year exposure to 0.5 ppm ozone resulted in atrophy of olfactory epithelium of female mice. At 1 ppm atrophy was also observed in male mice (Herbert et al., 1996). Understandably, there are no histopathological in vivo-studies investigating possible effects of ozone on the human olfactory epithelium. To our knowledge, there are also no ex vivo-examinations of human olfactory mucus following ozone exposure. A previous study on the effect of ozone on human olfactory functioning suggested an increase of butanol detection thresholds following a four hour exposure to 0.4 ppm (Prah and Benignus, 1979). Given the high concentration and the number of eight subjects, a need for further studies featuring lower exposures has been identified (Ajmani et al., 2016a). We had the opportunity to investigate further possible effects on human olfaction in the context of an unpublished feasibility study not related to rhinological parameters. Possible acute effects were expected to take place more in the (peripheral) olfactory mucosa than in the central olfactory pathways. The site of action is relevant, because different types of olfactory tests seem to address different portions of olfactory processing. Threshold tests are thought to reflect peripheral aspects of olfactory function to a stronger degree (Hummel and Welge-Lüssen, 2006). The n-butanol threshold test from Sniffin’ Sticks test set is validated and has been proven sensitive for acute effects of irritants (Hummel et al., 2007; Muttray et al., 2013; Muttray et al., 2004; Ottaviano et al., 2012). Our main research question was whether a two hour exposure to 0.2 ppm ozone caused an olfactory threshold shift.

3

In two experimental studies with organic solvents, interleukins IL-1ß and IL-8 in nasal secretions were found to be sensitive indicators for subclinical irritating effects (Mann et al., 2002; Muttray et al., 1999). These are relevant mediators, among others, for inflammation of the respiratory tract (Devalia and Davies, 1993; Leikauf et al., 1995). Another question was therefore whether possible effects of ozone on the olfactory threshold were associated with increases in concentrations in two proinflammatory cytokines in nasal secretions, stemming from the nasal respiratory epithelium.

2. Methods 2.1. Experimental design A concentration of 0.2 ppm ozone was chosen because it is relevant from an environmental medical point of view. At a lower concentration, the chances of discovering possible effects would have been lower. Ozone concentrations are highest during summer. Hence, to minimize possible ozone contact before the experiment, the study was conducted in the winter season. Due to flu-like infections of subjects, one experimental day had to be postponed to the beginning of April, however. To reduce the possible influence of airborne environmental pollutants, all experiments were performed on days without smog, fog, or atmospheric inversion. The weather forecast was used to schedule the experimental days. In all cases, the prediction was corroborated by measurements of environmental pollutants (Table 1). The measuring station “Mainz-Zitadelle” is nearest to the research lab. The measuring station is representative for an urban area with traffic and located downtown. In contrast, the lab is located on a small hill and is considerably more ventilated, resulting in a dilution of pollutants. All measured values of NO2 were clearly below the German 1-hour limit, which is set at 200 µg/m³. Thirty-one healthy male subjects were exposed once to ozone or sham (filtered air) for two hours according to a parallel group design. This design was chosen to avoid possible residual effects (Altman, 1991). Subjects were assigned randomly to the exposure or control group. Based on studies with organic solvents (Muttray et al., 2013; Muttray et al., 2004), the sample size seemed sufficient to answer the main question (i.e. a possible effect on olfactory functioning), especially since ozone is a strong oxidant and has a considerably stronger irritating effect on the mucous membranes. Ozone has a strong and pungent odor. An effective blinding would have demanded a strong and also irritating odorant. In this case, effects of the masking substance on target variables would have been possible. If a masking substance had also been used, additional experiments would have been necessary to investigate its effects, including possible combination effects on the target values. This expenditure could not be achieved with limited funding. Therefore, we did not attempt to blind subjects.

4

2.2. Subjects Thirty-one Caucasian men were recruited by means of posters. Participants were on no medication. A screening to exclude possible diseases included occupational and past-medical history, physical examination, stress electrocardiogram, body plethysmography including an unspecific provocation with methacholin, blood count, determination of -glutamyl transpeptidase, sedimentation rate, and urine analysis with a test strip (Combur 10®, Roche). Current nasal or paranasal sinus diseases were examined by history and nasal endoscopy. Olfactory functioning was tested with the Sniffin’ Sticks test to exclude those with olfactory impairment (Hummel et al., 1997). Both TDI scores (i.e. the sum of scores obtained for threshold, discrimination and identification measures) and the scores of the n-butanol threshold test were within the normal range of the corresponding age group (Hummel et al., 2007). Another purpose of the determination of the threshold for nbutanol was to familiarize the subjects with the method with respect to the forthcoming experiments. All subjects were healthy. After three current smokers were excluded (for cotinine levels see chapter 2.3.), the exposed group included 15 and the control group 13 subjects. All subjects exposed to ozone were lifelong nonsmokers. This also applied to the control subjects with the exception of one subject who stopped smoking three years ago. The median age was 23 years (20-28) and 24.5 years (20-27), respectively. All the subjects were students except for one who was still waiting for a place at university. The study was performed in accordance with the ethical principles of the Declaration of Helsinki and its latest amendments. The protocol was approved by the local ethics committee (“Ethik-Kommission bei der Landesärztekammer RheinlandPfalz“). It included pulmonary function tests for the purpose of the subjects’ safety. Written informed consent was obtained from every test person. Subjects were paid for participation.

2.3. Previous exposure to airborne pollutants Smoking was an exclusion criterion, as it may impair olfactory functioning (Frye et al., 1990; Gudziol et al., 2013). Therefore, cotinine in urine was determined by gas chromatography/mass spectrometry after liquid-liquid extraction (Müller, 2006) in order to ensure that all subjects were nonsmokers. Analyses indicated that three subjects were active smokers (cotinine levels 184, 348, and 863 µg/l). These persons were subsequently excluded from analyses. Thus, the exposed group included 15 and the control group 13 subjects. Their respective average cotinine levels were 6.2 ± 7.9 (SD) µg/l and 9.3 ± 9.8 µg/l. All values were clearly under the cut-off value of 49.7 µg/l (Jarvis et al., 1987). To the best of our knowledge, they did not use

5

tetrahydrocannabinol or other drugs. Subjects were not engaged in jobs which might be associated with an exposure to neurotoxic substances or irritants such as welding fumes. They had no hobbies associated with an irritant or toxic exposure. For further information on previous airborne environmental exposures, we asked the competent authorities of the Federal States Hesse and Rhineland-Palatinate for measured values of airborne pollutants. The measuring stations most representative for the subjects’ primary residences were selected. Immission spectra of O3, NO2, PM10 and SO2 were provided. Unfortunately, there are no representative measurements of exposure to PM 2.5, which is due to the lack of measurement sites in the region. The evaluation of the other pollutants was restricted to the previous year, since many subjects had changed their primary residencies in the past. Table 2 provides an overview of the average annual immissions of O3, NO2 and PM10. The information threshold of 180 µg/m³ (0.09 ppm) O3 average per hour was exceeded at some measuring stations during summer of the previous year. The median number of exceedances related to the different subjects was 0 for both groups (3rd quartile 17, maximum 24, in each group). The target value for ozone (120 µg/m³, 0.06 ppm) is related to a 3-year period, which is not covered by our data. The average annual limit for NO2 is fixed at 40 µg/m³. It was exceeded at two measuring stations, one related to an ozone-exposed subject, the other to a control person (see maximum values given in Table 2). The average hourly limit for NO2 (200 µg/m³) was never exceeded. The annual limit for PM10 (40 µg/m³) was complied with (Table 2). The hourly PM10 limit is fixed at 50 µg/m³. This limit may be exceeded 35 times per year. The number of exceedances was well below this limit in all subjects but one ozone-exposed person. He lived near a measuring station downtown. In this case, the limit was exceeded 45 times. No representative values of NO and PM2.5 are available, as only very few measuring stations have recorded these pollutants. Average annual sulphur dioxide concentrations were low (range 2.3 – 4.5 µg/m³). Both the average hourly and the daily concentrations were well below the respective limits (350 µg/m³ and 125 µg/m³) No representative values of ammonia were available. The main source of this pollutant is animal farming. No subject lived in the neighborhood of a farm. Ozone concentration in ambient air outdoors near the lab, measured before the experiments, ranged from 0.0135 to 0.03 ppm.

2.4. Experimental exposure Exposure was carried out in an 18-m3 chamber. Exposure time was two hours. The chamber was supplied with carbon filtered outdoor air. Ozone was produced from ambient air with ozone generators (COM-SD.30, Anseros Klaus Nonnenmacher GmbH, Tübingen, Germany) and led into the exposure chamber. To achieve a stable ozone concentration in the empty chamber, two ozone generators were sufficient. However, ozone is absorbed

6

by clothes (Henschler et al., 1960). Preliminary experiments indicated four ozone generators were needed to achieve stable concentrations when subjects were in the exposure chamber. Ozone concentration was measured in the chamber every five minutes (U.V. Photometric Ambient O3 Analyzer/Calibrator, Thermo Environmental Instruments, Franklin, Massachusetts). Ozone supply was adjusted, if necessary. Mean ozone concentration of the exposure group was 0.21 ppm (± 0.005 SD). Maximum ozone concentration during the control experiments was 0.014 ppm. Mean temperature was 22.0 ºC (± 1.0 SD) and 21.8 ºC (± 0.4 SD) for ozone and sham exposure, respectively. Humidity was 43.4 % (± 1.8 SD) and 44.2 % (± 1.1 SD), respectively. The air exchange rate was 6 times per hour.

2.5. Experimental procedure Exposure was conducted at least seven days after the preliminary medical examination. Subjects were advised to avoid excessive outdoor activities prior to their exposure. Each experimental day started at 8:00 a.m. with the first subject. Up to two other subjects were tested consecutively, with intervals of 75 min in between. Fig. 1 shows the study timeline.

Fig. 1: Timeline for a test subject Measures of dependent variables of this study are in bold type. SPES: Questionnaire addressing various acute symptoms derived from the Swedish Performance Evaluation System. h: hours.

Upon arrival, each subject was medically assessed. A general questionnaire (e.g. intermediate health complaints) as well as a questionnaire addressing various acute symptoms were filled out (Seeber et al., 2002). The olfactory threshold was measured in a neighbored quiet and well-ventilated room. A urine sample was taken for the determination of cotinine concentration. Afterwards, tests not related to the target variables of this study were performed (Fig. 1). After a break, the subject entered the exposure chamber and was exposed to ozone or filtered

7

air for 2 hours, respectively. The questionnaire related to acute symptoms was completed again upon entrance, after one hour and at the end of exposure. Fifteen and 70 min after the beginning of exposure, all subjects had to cycle on an ergometer maintaining 100 W of power for 15 min to enhance pulmonal and also nasal ventilation (Bennett et al., 2003). Subjects were continuously observed. They could read, communicate with each other, eat and drink in the exposure chamber. We made sure that subjects did not ingest beverages and food that might impair olfaction or irritate mucous membranes. As the measurement of the olfactory threshold is a psychophysiological method, the results do also depend on psychological factors. Therefore, sensations such as hunger and thirst, which might impair the subjects’ concentration during olfactory tests, should be prevented. Subjects were not allowed to sleep. After leaving the exposure chamber, pulmonary function was tested to monitor subjects. The olfactory function test is a psychophysiological examination. In order to minimize theoretically possible influences of the previous examinations (blood sampling and pulmonary function test), the subjects took a monitored break of 20 minutes before the olfactory test began. Olfactory threshold was measured 70 min after exposure. Afterwards, nasal secretions were taken. Finally, subjects were asked by questionnaire to rate the exposure level to ozone as explained in the next paragraph. The questionnaire was not presented immediately after exposure to avoid possible carryover effects from reflecting on the questions on olfactory testing as well as filling out the questionnaire on subjective symptoms.

2.6. Subjective ratings of exposure level A self-assessment questionnaire was used to distinguish whether subjects were aware of the exposure condition (ozone vs. filtered air). The wording was: “How strongly did you perceive today’s ozone concentration in the chamber?” The question has been formulated broadly, because subjects should consider all possible effects of exposure, e.g. irritating effects, and not only the kind and the perceived intensity of odor. They rated their perceived level of exposure on a visual analogue scale from 0 (no ozone detectable) to 100 (very high concentration) at the end of the experimental day. Additionally, they were asked to explain their judgments.

2.7. Assessment of acute symptoms The questionnaire addressing various acute symptoms was derived from the Swedish Performance Evaluation System (SPES) (Iregren et al., 1996). The extended version (Seeber et al., 2002) comprises 29 items with an ordinal scale of 0 (equals no complaints at all) to 5 (equals maximum discomfort, pain or symptoms). The subjects were familiarized with the questionnaire before the experimental day. The whole questionnaire was

8

completed before exposure, shortly after entering the exposure chamber, after one hour exposure and at the end of exposure (Fig. 1). Regarding the scope of this study, the evaluation of scores focusses on items related to the nose. i.e. “irritation to the nose”, ”itching nose”, ”dry nose”, ”running nose”, and ”burning nose”. In addition, the scores related to symptoms of the noses were grouped and their means related to the different time points were calculated (van Thriel et al., 2003). Other symptoms include “sensation of bad smell” and “coughing”.

2.8. Rhinological and laboratory examinations The olfactory threshold for n-butanol (range of scale 1 - 16) was assessed with the Sniffin’ Sticks test (Hummel et al., 1997). The sticks were new and kept in climate-controlled storage (Denzer et al., 2014). Subjects were instructed to abstain from sharply flavored food, onions, garlic, perfume, and shaving lotions preceding the day of the experiment. They did not eat or drink within at least 20 minutes before olfactory testing. Subjects were blindfolded with a sleeping mask. They received no feedback on their decisions. Olfactory tests were performed using both nostrils. Nasal secretions were collected for 15 minutes with absorbent foam rubber samplers (Klimek and Rasp, 1999). This method has proven sensitive in previous studies (Mann et al., 2002; Muttray et al., 1999). Samples were taken both during the pre-trial proceedings and one and a half hours after the end of exposure. The interval between the samplings was at least seven days to prevent possible residual effects. Secretions were centrifuged and deep frozen at –80°C. Interleukin 1β (IL-1ß) was analyzed with a commercial ELISA QuantiGlo® human IL-1ß (R&D Systems, Wiesbaden, FRG). For quantitative determination of interleukin 8 (IL-8) in nasal secretions a sandwich enzyme immunoassay with absorption-based detection (DuoSet® ELISA Development System human IL-8, R&D Systems) was used. Results were expressed as the means of duplicate measurements. All laboratory analyses were performed blindly.

2.9. Statistics The applicability of a normal distribution assumption was examined using histograms and skewness. Normal distribution was assumed if the histogram showed only one peak and the skewness was between -1 and 1. For visual presentation, olfactory threshold values before exposure were subtracted from those after exposure in both exposure groups. With regard to previous research (Prah and Benignus, 1979), it was assumed that an exposure to ozone would impair olfactory functioning. Scores of olfactory thresholds were logarithmized (base of 10) to normalize data. Then, a mixed-design analysis of variance (ANOVA) was performed to find out how

9

between-subject effects of exposure (ozone vs. sham) and within-subject effects of time (before vs. after exposure) impacted the log transformed olfactory threshold score. Concentrations of interleukins IL-1ß and IL-8 in nasal secretions were measured during the preliminary examination and at the experimental day after exposure. Values obtained at the initial examination were subtracted from those obtained after exposure. As ozone irritates mucous membranes and other irritants increased concentrations of interleukins in nasal secretions (Mann et al., 2002; Muttray et al., 1999), the null hypothesis was that the differences of interleukin concentrations after ozone exposure equalled or were lower than the respective differences after sham exposure. The alternative hypothesis was that the differences of interleukin concentrations after ozone exposure were higher. The global significance level was set at p < 0.05. In total, three hypotheses (possible effects on olfactory threshold and secretion of IL-1β and IL-8) were tested. The local p levels were adjusted for multiple testing with the Bonferroni procedure (i.e. p < 0.05/3). For comparison of questionnaire scores related to nasal symptoms, values before exposure were subtracted from those during or after exposure. The respective questionnaire scores (ratings of exposure and subjective symptoms) of the groups exposed to ozone and sham were compared exploratively. Respective p values (Mann-Whitney U test) are given for descriptive reasons only. Analyses were performed with SPSS (IBM SPSS Statistics for Windows, Version 19.0. Armonk, NY). For descriptive statistics, measures of central tendency (mean, median) and scatter (standard deviation, quartiles) are shown depending on the kind of the respective distributions. Alternative calculations with the exclusion of the subject with the highest exposure to PM 10 yielded similar results (data not shown).

3. Results 3.1. Rhinological examinations 3.1.1. Olfactory functioning The median olfactory threshold scores for n-butanol before exposure was 8.75 (1st quartile 8.0, 3rd quartile 10.75) for the ozone group and 8.0 (7.5, 8.88) for the control group. Thus, there was no relevant difference between the initial values of the two groups. The ANOVA revealed a main effect for ozone exposure (F (1, 26) = 27.6, p= 0.0002, η2 = 0.59), for time (F (1, 26) = 13.5, p= 0.0011, η 2 = 0.34), as well an interaction effect between exposure and time (F (1, 26) = 22.8, p= 0.0006, η2 = 0.47) on the olfactory threshold for n-butanol. The interaction effect represents the combined effects of factors exposure and time on the dependent measure

10

olfactory threshold. For visualization, all individual values are shown and the differences of the post and respective pre thresholds were calculated for both groups (Fig. 2 and 3).

A

B

Fig. 2. Effect of ozone exposure on the olfactory threshold for n-butanol – individual results A: Subjects exposed to ozone (n=15). B: Subjects exposed to filtered air (n=13). Gray bars indicate the olfactory thresholds before exposure, black bars those afterwards. The higher the score the better olfaction.

11

Fig. 3. Effect of ozone exposure on the olfactory threshold for n-butanol – differences on group basis Ozone group: n=15, control group n=13. ANOVA, p for exposure effect. Boxplots: The 1st quartile value is at the lower horizontal line of the box, the 3rd quartile value is at the upper horizontal line, the thick line in the box represents the median. The whiskers (the lines that extend out the top and bottom of the box) represent the highest and lowest values that are not outliers. Outliers are values between 1.5 and 3 times the interquartile range and represented by circles beyond the whiskers.

3.1.2. Inflammatory factors Concentration of IL-1β in nasal secretions did not increase after exposure to ozone when compared to sham (median difference of concentrations post – pre 20 pg/ml, 1st quartile -43 pg/ml, 3rd quartile 31 pg/ml; respective control values were -13, -28.5 and -2.5 pg/ml). Exposure to ozone did likewise not elevate the concentration of IL-8 in nasal secretions. The differences of the concentrations (post – pre) were -342 pg/ml (± 2114 pg/ml (SD)) and -245 pg/ml (± 1177 pg/ml) for ozone and sham exposure, respectively.

3.2. Ratings of exposure On group basis, ozone exposed subjects rated exposure considerably higher than control subjects (Fig. 2). Therefore, the study cannot be considered blind. The justifications of the ozone-exposed subjects were mainly related to irritating effects, but also to odor. They described the smell of ozone in different ways, i.e. like chlorine in a swimming pool, strange (different German wordings), and unpleasant.

12

Fig. 4. Perceived level of exposure Subjects rated the perceived level of exposure on a visual analogue scale (0100). Ozone group: n=15, control group n=13. Mann-Whitney U test. Boxplots: The 1st quartile value is at the lower horizontal line of the box, the 3rd quartile value is at the upper horizontal line, the thick line in the box represents the median. The whiskers (the lines that extend out the top and bottom of the box) represent the highest and lowest values that are not outliers. Outliers are values between 1.5 and 3 times the interquartile range and represented by circles beyond the whiskers.

3.3. Subjective symptoms No clear-cut irritating symptoms related to the nose were observed (Table 3). Accordingly, the composite scores for the five irritating nasal symptoms revealed no differences between both groups at the various time points. When entering and before leaving the chamber, the median score for the “sensation of bad smell“ was slightly increased in the subjects exposed to ozone (Table 3). At the end of exposure, the score for “cough“was increased in ozone-exposed subjects (Table 3 and Fig. 5). All these measures are self-reported. Following ozone exposure, some subjects had to cough during deep inspiration in the course of pulmonary function testing. No subjects had to cough during pulmonary function testing before exposure or after sham exposure.

13

Fig. 5. Individual scores of the item cough before and during exposure All subjects completed the questionnaire before exposure ( ), upon entrance in the exposure chamber (), after one hour of exposure ( ), and at the end of exposure (2 hours ). The ordinal scale ranges from 0 to 5. Many measurements were zero. For better visualization the bars start below the abscissa. Numbers below the bars indicate the individual subjects.

4. Discussion A concentration of 0.2 ppm ozone can be found outdoors and at workplaces under unfavorable conditions. To the best of our knowledge, this is the first study showing an acute effect of 0.2 ppm ozone on the human olfactory threshold.

4.1. Possible modes of action The physiological basis of the olfactory threshold shift observed in the present study is subject of further research. However, different possible modes of action shall be examined in more detail here. The olfactory epithelium is covered by a thin layer of aqueous mucus containing antioxidants. Ozone reacts with these antioxidants. When the antioxidative capacity of the mucus is reached, ozone can react with different

14

compounds in the olfactory mucus. The olfactory cilia which form a dense network in the mucus (Dennis et al., 2015). Different lipids of cilia contain carbon-carbon double bonds (Lobasso et al., 2010). Ozone is known to react rapidly with such double bonds (Crigee mechanism) (Bromberg, 2016). Ozone can also react with other components of the olfactory mucus such as proteins and glycoproteins. Proteins are part of the odorant receptors located on the cilia (Heydel et al., 2013). Thus, it is conceivable that ozone and/or its reaction products might have impaired the function of the olfactory receptors. Another mode of action might also play a role. One hypothesis is that so-called odorant binding proteins transport small hydrophobic molecules to the olfactory receptors (Silva Teixeira et al., 2016). According to its octanol/water partition coefficient, n-butanol is classified as hydrophobic (Hazardous Substances Data Bank (HSDB), 2018). If the hypothesis were confirmed, an impairment of the transport of n-butanol would be a possible mode of action. As reaction products, various free radicals might react with cell structures and induce inflammation (Bromberg, 2016; Croze and Zimmer, 2018; Kelly et al., 1995; Yan et al., 2016). A result might be an increase of the mucous membranes’ permeability when the ozone concentration is high enough (Nikasinovic et al., 2003), possibly resulting in changes of the olfactory mucus. Conditions that effect its ion composition, viscosity, or the proteins within, can alter the ability to perceive volatile chemicals (Rawson, 1999). An examination of the olfactory cleft mucus (Debat et al., 2007; Wu et al., 2018) could be one way to check if ozone causes inflammation in the olfactory mucosa. Interactions between the trigeminal and the olfactory system have been observed (Brand, 2006). In humans, the simultaneous intranasal application of the trigeminal irritant CO2 attenuated the olfactory response to the odorant phenylethyl alcohol (Daiber et al., 2013). This raises the question whether ozone has irritated trigeminal receptors in the nasal mucosa. In this study, two hours of exposure to 0.2 ppm ozone did not cause any clinical symptoms suggesting irritation to the respective trigeminal receptors. This result is in line with other studies (Henschler et al., 1960; Higgins et al., 1979; Jörres et al., 1996; Jörres et al., 2000; Scannell et al., 1996). Very high concentrations of ozone can cause irritation of the trigeminal nerve both in humans and rats (Kulle and Cooper, 1975; Nasr, 1971; Sanderson et al., 1999). The question of whether possible trigeminal irritation in our study was of a certain relevance for olfactory performance cannot be answered at this time. Possibly, the measurement of neuropeptides such as substance P in nasal secretions (Mosimann et al., 1993; Schierhorn et al., 2002) might provide further insights. We did not find effects on the proinflammatory cytokines IL-1ß and IL-8 stemming from the nasal respiratory mucosa. This missing increase does not support the inflammation hypothesis. On the other hand, it cannot be

15

ruled out that a longer interval between exposure and sampling nasal secretions would have increased the chances to detect inflammatory changes (Leikauf et al., 1995). Consequently, mediators might not have captured what was going on in the mucosa. Another possible mode of action are effects on the central nervous system (CNS). Ozone is a very powerful oxidant. It induces inflammation not only in but also outside the airways via local generation of various secondary mediators (Erickson et al., 2017). Oxidative damage and inflammation may expand from the respiratory system to other organs, including the brain (Croze and Zimmer, 2018; Devlin et al., 2012; Erickson et al., 2017; Laskin et al., 1994). In rat studies, acute exposure to ozone caused oxidative stress in some regions of the brain in comparatively high doses (i.e. 0.4–1.5 ppm for 4 h, or 1.0 ppm for 1–9 h) (Croze and Zimmer, 2018; Mokoena et al., 2010). However, no increase in lipid peroxidation was observed after a single four-hour exposure to 0.25 ppm ozone (Mokoena et al., 2010). Cytological and ultrastructural changes to the olfactory bulb were observed in rats exposed to 1-1.5 ppm ozone four hours (Colin-Barenque et al., 1999), attributed to oxidative stress. In contrast, our test subjects were exposed to 0.2 ppm ozone for 2 hours. The question is whether the results of the experiments with rats can be transferred to our study because exposure was considerably lower. However, higher doses of ozone appear to be required in rats in order to achieve results comparable to those obtained in humans. Resting rats had to be exposed to 2 ppm 18O3 in order to achieve a cell dose in bronchoalveolar lavage fluid similar to exercising humans exposed to 0.4 ppm (Hatch et al., 2013; Hatch et al., 1994). A controlled acute exposure to 0.3 ppm ozone induced changes in markers of vascular inflammation and responses in the human cardiovascular system (Devlin et al., 2012). Taken together, it seems possible that neuroinflammatory changes in the CNS of our subjects might have contributed to an increase in the olfactory threshold. Another mechanism, namely adaptation, was discussed in a previous study (Prah and Benignus, 1979). Eight subjects were exposed to 0.4 ppm ozone for 4 h on four consecutive days. The “sensitivity” to n-butanol was decreased after the first ozone exposure. On the subsequent days, the effect was attenuated. By the end of the fourth day of exposure, olfactory thresholds returned to baseline. Adaptation means that a repeated or prolonged exposure to an odorant decreases the sensitivity to that specific odorant (Dalton, 2000). However, ozone and nbutanol are different chemical compounds. Therefore, adaptation can be excluded. Instead, the possibility of cross-adaptation has to be discussed. Olfactory receptors exhibit a combinatorial response to odorant molecules. A single odor elicits a response from multiple receptors and a single receptor responds to multiple odorants (Araneda et al., 2000; Malnic et al., 1999). Thus, every odorant is thought to have a unique combination of

16

responses from several receptors (Malnic et al., 1999). An overlap between chemicals is more probable if they are sterically similar and have the same functional group (Malnic et al., 1999; Pierce et al., 1996; Zhao et al., 1998), e.g., an alcohol group. However, the chemical structures of ozone and n-butanol are different. Olfactory adaptation can also occur with chemicals of dissimilar structure if they possess similar odor characteristics (Cometto-Muniz and Cain, 1995; Gottfried et al., 2006; Todrank et al., 1991). As the descriptions of the test subjects and the authors’ perceptions show, the smell of ozone is hard to describe. The wording “electric smell” (Henschler et al., 1960) is probably related to the formation of ozone in connection with electrical arcing. In any case, the odor of ozone is quite different from the mildly alcoholic odor of n-butanol. For these reasons, crossadaptation between ozone and n-butanol is very unlikely.

4.2 Reversibility of the olfactory threshold shift In this study, it was not possible to investigate the olfactory threshold over a longer period of time. The study of Prah and Benignus (1979) suggests that the acute effect of ozone on olfaction is reversible. An alternative explanation for the temporal progression of olfactory functioning (Prah and Benignus, 1979) might be a training effect.

4.3 Effect size Our study found that the median olfactory threshold for n-butanol in the ozone group was 1.25 higher when the intraindividual reference values were considered (Fig. 3). A higher score means better olfactory performance. We regard this as a weak effect. For comparison, the effect size in this study was more than twice as high as that of smokers after smoking a cigarette (decrease of the average score from 7.26 to 6.80) (Graul, 2014). As for the chronic effects of cigarette smoking, the mean threshold for n-butanol was 1.2 higher in smokers than in nonsmokers (scores 7.3 vs. 8.5) (Katotomichelakis et al., 2007).

4.4 Limitations of the study Experiments were not performed on days with fog or thermal inversions. Nevertheless, it was not possible to standardize pre-exposures to airborne pollutants with respect to subjects’ primary residencies. Recently, an association between exposure to PM 2.5 and dysosmia has been reported (Ajmani et al., 2016b). Unfortunately, there are no representative measurements of PM 2.5 for the residences of our test subjects. To minimize possible effects of different pre-exposures, a pre-post-measurement design was chosen. No drug tests were performed.

17

This might be a limitation, even if we had no suspicion of drug abuse. We did not use an odorous substance to mask the odor of ozone, because effects on target variables could not be excluded. The question is whether the missing blinding could have biased our study and, if so, to what extent. Especially with subjective endpoints there is the possibility of a bias if no blinding was performed or if it was incomplete (Miller and Stewart, 2011). In our study, subjects’ condition, which was investigated by means of questionnaires, represents a subjective endpoint. Psychophysiological threshold tests, in which the subject is asked what he has perceived, also have a subjective endpoint. Because the examiner cannot control whether the result is correct or not (Ehrenstein and Ehrenstein, 1999). However, we have chosen a forced-choice method to determine the olfactory threshold. A forced-choice method provides a more objective approach (Ehrenstein and Ehrenstein, 1999). The subject is required to make a positive response on every trial – regardless of whether he smelled the stimulus. In addition, observation of the test persons showed no evidence of insufficient cooperation. Subjects could communicate. Even if ozone-exposed persons showed no overt signs of irritation, communication cannot be excluded as a source of error with certainty. Our subjects were healthy young men, which are not representative for the whole population. The effect may only be generalizable to men, as there are differences in olfactory function between males and females (Doty et al., 1984; Hummel et al., 2007).

4.5 Effects of ambient air pollutants including ozone on olfaction This study showed an acute effect of ozone on the olfactory threshold. Understandably, there are no studies that have investigated the effect of chronic exposure to ozone alone on the human olfactory system. Decrements in smell function are common in areas highly polluted with mixtures of pollutants including ozone (Ajmani et al., 2016a; Calderon-Garciduenas, 2015; Hudson et al., 2006). In particular, concentrations of PM10, PM2.5 and ozone were very high in Mexico City (Ajmani et al., 2016a). Pronounced pathological changes in the nasal respiratory epithelium, a marked decrease in olfactory neurons, pronounced changes in Bowman’s glands, and pathologic changes within the olfactory bulbs were observed (Calderon-Garciduenas, 2015). Mexico City residents, but not controls, exhibited neuronal periglomerular accumulation of particulate material and immunoreactivity to alpha-synuclein, amyloid beta Aβ42, and hyperphosphorilated tau in the olfactory bulbs (Calderon-Garciduenas, 2015). This result speaks for the pathogenetic significance of particular matters. In the case of a mixed exposure, it is not possible to indicate the exact pathogenetic significance of the ozone exposure. In our study, oxidative stress and inflammation, either locally or in the CNS, could have caused an increase in olfactory threshold. In this respect, our result is compatible with the impairment of the olfactory

18

ability of people who were highly exposed to air pollutants (Calderon-Garciduenas, 2015; CalderonGarciduenas et al., 2015). However, our study cannot contribute to further clarification of the pathogenesis.

Acknowledgements This study was supported in part by a grant from the Ministry of Environment and Forests of the German Regional State Rhineland-Palatinate and the Johannes Gutenberg University Mainz.

Conclusion This is the first study showing a clear impairment of human olfactory functioning following an acute exposure to 0.2 ppm ozone. This concentration is relevant with regard to actual environmental levels.

Conflicts of interest None.

Funding This work was supported in part by a grant from the Ministry of Environment and Forests of the German regional state Rhineland-Palatinate and the Johannes Gutenberg University Mainz. The funders had no role in study design, in the collection, data analysis and interpretation, in the writing of the manuscript, and in the decision to submit the manuscript for publication.

Approval by the local ethics committee The protocol was approved by the local ethics committee (“Ethik-Kommission bei der Landesärztekammer Rheinland-Pfalz“, http://www.laek-rlp.de/ethik/index.php).

References Achcar, J. A., et al., 2013. Some dependence models for the time between ozone exceedances in Mexico City. Environ Model Assess. 18, 259-270. Ajmani, G. S., et al., 2016a. Effects of ambient air pollution exposure on olfaction: a review. Environ Health Perspect. 124, 1683-1693.

19

Ajmani, G. S., et al., 2016b. Fine particulate matter exposure and olfactory dysfunction among urban-dwelling older US adults. Environ Res. 151, 797-803. Altman, D. G., 1991. Practical statistics for medical research. CRC Press, Boca Raton. Araneda, R. C., et al., 2000. The molecular receptive range of an odorant receptor. Nat Neurosci. 3, 1248-55. Banta, R. M., et al., 2011. Dependence of daily peak O3 concentrations near Houston, Texas on environmental factors: Wind speed, temperature, and boundary-layer depth. Atmospheric Environment. 45, 162-173. Bekö, G., et al., 2015. Impact of cabin ozone concentrations on passenger reported symptoms in commercial aircraft. PLoS One. 10, e0128454. Bennett, W. D., et al., 2003. Nasal contribution to breathing with exercise: effect of race and gender. J Appl Physiol. 95, 497-503. Brand, G., 2006. Olfactory/trigeminal interactions in nasal chemoreception. Neurosci Biobehav Rev. 30, 90817. Bromberg, P. A., 2016. Mechanisms of the acute effects of inhaled ozone in humans. Biochim Biophys Acta. 1860, 2771-81. Calderon-Garciduenas, L., Influence of toxins on olfactory function and their potential association with neurodegenerative disease. In: R. L. Doty, (Ed.), Handbook of olfaction and gustation. John Wiley & Sons, Hoboken, 2015, pp. 485-509. Calderon-Garciduenas, L., et al., 2015. Megacities air pollution problems: Mexico City Metropolitan Area critical issues on the central nervous system pediatric impact. Environ Res. 137, 157-69. Cardis, D., et al., 2007. Ozone in the laundry industry - practical experiences in the United Kingdom. Ozone Sci Eng. 29, 85-99. Carey, S. A., et al., 2011. Persistent rhinitis and epithelial remodeling induced by cyclic ozone exposure in the nasal airways of infant monkeys. Am J Physiol Lung Cell Mol Physiol. 300, L242-L254. Cercelaru, C., et al., 2016. Odour causing discomfort and thier impact on human health. J Environ Protection Ecology. 17, 427-434. Chelani, A., 2013. Study of extreme CO, NO2 and O3 concentrations at traffic site in Delhi: statistical persistence analysis and source identification. Aerosol Air Quality Res. 13, 377-384. Colin-Barenque, L., et al., 1999. Morphologic alteration of the olfactory bulb after acute ozone exposure in rats. Neurosci Lett. 274, 1-4.

20

Cometto-Muniz, J. E., Cain, W. S., Olfactory adaptation. In: R. L. Doty, (Ed.), Handbook of olfaction and gustation. Marcel Dekker, New York, 1995, pp. 257-281. Croze, M. L., Zimmer, L., 2018. Ozone atmospheric pollution and Alzheimer's disease: From epidemiological facts to molecular mechanisms. J Alzheimers Dis. 62, 503-522. Daiber, P., et al., 2013. Neuropeptide receptors provide a signalling pathway for trigeminal modulation of olfactory transduction. Eur J Neurosci. 37, 572-82. Dalton, P., 2000. Psychophysical and behavioral characteristics of olfactory adaptation. Chem Senses. 25, 48792. Debat, H., et al., 2007. Identification of human olfactory cleft mucus proteins using proteomic analysis. J Proteome Res. 6, 1985-96. Dennis, J. C., et al., Development, morphology, and functional anatomy of the olfactory epithelium. In: R. L. Doty, (Ed.), Handbook of olfaction and gustation. John Wiley & Sons, Hoboken, 2015, pp. 93-108. Dennis, J. H., et al., 2002. Control of occupational exposure to hexavalent chromium and ozone in tubular wire arc-welding processes by replacement of potassium by lithium or by addition of zinc. Ann Occup Hyg. 46, 3342. Denzer, M. Y., et al., 2014. Quantitative validation of the n-butanol Sniffin’ Sticks threshold pens. Chemosens Percept. 7, 91-101. Devalia, J. L., Davies, R. J., 1993. Airway epithelial cells and mediators of inflammation. Respir Med. 87, 4058. Devlin, R. B., et al., 2012. Controlled exposure of healthy young volunteers to ozone causes cardiovascular effects. Circulation. 126, 104-11. Doty, R., et al., 1984. Smell identification ability: changes with age. Science. 226, 1441-1443. Ehrenstein, W. H., Ehrenstein, A., Psychophysical methods. In: U. Windhorst, H. Johansson, Eds.), Modern techniques in neuroscience research. Springer, Berlin, 1999, pp. 1211-1241. Environmental Protection Agency, Integrated science assessment for ozone and related photochemical oxidants. EPA/600/R-10/076F. Triangle Park, 2013. https://cfpub.epa.gov/ncea/isa/recordisplay.cfm?deid=247492, assessed at 20-April-2018. Environmental Protection Agency, National Ambient Air Quality Standards (NAAQS) for Ozone. Vol. 80. Federal Register, Triangle Park, 2015, pp. 65292-65684. https://www.gpo.gov/fdsys/pkg/FR-2015-1026/pdf/2015-26594.pdf, assessed at 20-April-2018.

21

Erickson, M. A., et al., 2017. Serum amyloid A: an ozone-induced circulating factor with potentially important functions in the lung-brain axis. Faseb J. 31, 3950-3965. European Commission, Air quality standards. 2016. http://ec.europa.eu/environment/air/quality/standards.htm, assessed at 20-April-2018. Evers, U., Nowak, D., 2006. Erkrankungen durch Emissionen aus Laserdruckern und Kopiergeräten? Gefahrstoffe Reinhaltung Luft 66, 203-210. Fachausschuss Metall- und Oberflächenbehandlung, Exposure to ozone during welding and allied processes. Expert Committee Information Sheet No. 041. Draft 02/2009. Deutsche Gesetzliche Unfallversicherung, 2009. https://www.bghm.de/fileadmin/user_upload/Arbeitsschuetzer/Praxishilfen/FachbereichsInformationsblaetter/041_MO_E2009-02_Ozone-Exposure.pdf, assessed at 20-April-2018. Frye, R., et al., 1990. Dose-related effects of cigarette smoking on olfactory function. J Am Med Assoc. 263, 1233-1236. Gottfried, J. A., et al., 2006. Dissociable codes of odor quality and odorant structure in human piriform cortex. Neuron. 49, 467-79. Graham, D. E., Koren, H. S., 1990. Biomarkers of inflammation in ozone-exposed humans. Comparison of the nasal and bronchoalveolar lavage. Am Rev Respir Dis. 142, 152-6. Graul, J., Einfluss des Rauchens auf das Riechvermögen des Menschen. Medizinische Fakultät. FriedrichSchiller-Universität Jena, 2014. http://d-nb.info/1048047091/34, assessed at 20-April-2018. Gudziol, H., et al., 2013. Ability of smelling is reduced reversibly by acute smoking and permanently by chronic smoking. Laryngorhinootologie. 92, 663-6. Harkema, J. R., et al., 1987. Response of the macaque nasal epithelium to ambient levels of ozone. A morphologic and morphometric study of the transitional and respiratory epithelium. Am J Pathol. 128, 29-44. Hatch, G. E., et al., 2013. Biomarkers of dose and effect of inhaled ozone in resting versus exercising Human subjects: Comparison with resting rats. Biomark Insights. 8, 53-67. Hatch, G. E., et al., 1994. Ozone dose and effect in humans and rats. A comparison using oxygen-18 labeling and bronchoalveolar lavage. Am J Respir Crit Care Med. 150, 676-83. Hazardous Substances Data Bank (HSDB), n-butanol. 2018. https://toxnet.nlm.nih.gov/newtoxnet/hsdb.htm, assessed at 20-April-2018.

22

Henschler, D., et al., 1960. Odor threshold of a few important irritant gases (sulfur dioxide, ozone, nitrogen dioxide) and observations in humans exposed to low concentrations Arch Gewerbepathol Gewerbehyg. 17, 547570. Herbert, R. A., et al., 1996. Two-year and lifetime toxicity and carcinogenicity studies of ozone in B6C3F1 mice. Toxicol Pathol. 24, 539-48. Heydel, J. M., et al., 2013. Odorant-binding proteins and xenobiotic metabolizing enzymes: implications in olfactory perireceptor events. Anat Rec (Hoboken). 296, 1333-45. Higgins, E. A., et al., Effects of ozone on exercising and sedentary adult men and women representative of the flight attendant population. Federal Aviaton Administration Oklahoma City OK Civil Aeromedical Institute, 1979. http://www.dtic.mil/dtic/tr/fulltext/u2/a080045.pdf, assessed at 20-April-2018. Hoffman, C. D., et al., 2004. Exposure to ozone gases in pulp mills and the onset of rhinitis. Scand J Work Environ Health. 30, 445-9. Hudson, R., et al., 2006. Effect of air pollution on olfactory function in residents of Mexico City. Chem Senses. 31, 79-85. Hummel, T., et al., 2007. Normative data for the "Sniffin' Sticks" including tests of odor identification, odor discrimination, and olfactory thresholds: an upgrade based on a group of more than 3,000 subjects. Eur Arch Otorhinolaryngol. 264, 237-43. Hummel, T., et al., 1997. "Sniffin' sticks": olfactory performance assessed by the combined testing of odor identification, odor discrimination and olfactory threshold. Chem Senses. 22, 39-52. Hummel, T., Welge-Lüssen, A., 2006. Assessment of olfactory function. Adv Otorhinolaryngol. 63, 84-98. Iregren, A., et al., 1996. SPES: a psychological test system to diagnose environmental hazards. Neurotoxicol Teratol. 18, 485-91. Jarvis, M. J., et al., 1987. Comparison of tests used to distinguish smokers from nonsmokers. Am J Public Health. 77, 1435-8. Jörres, R., et al., 1996. The effect of ozone exposure on allergen responsiveness in subjects with asthma or rhinitis. Am J Respir Crit Care Med. 153, 56-64. Jörres, R. A., et al., 2000. The effect of repeated ozone exposures on inflammatory markers in bronchoalveolar lavage fluid and mucosal biopsies. Am J Respir Crit Care Med. 161, 1855-61. Katotomichelakis, M., et al., 2007. Normative values of olfactory function testing using the 'Sniffin' Sticks'. Laryngoscope. 117, 114-20.

23

Kelly, F. J., et al., 1995. The free radical basis of air pollution: focus on ozone. Respir Med. 89, 647-56. Klimek, L., Rasp, G., 1999. Norm values for eosinophil cationic protein in nasal secretions: Influence of specimen collection. Clin Exp Allergy. 29, 367-374. Kulle, T. J., Cooper, G. P., 1975. Effects of formaldehyde and ozone on the trigeminal nasal sensory system. Arch Environ Health. 30, 237-43. Laskin, D. L., et al., 1994. Pulmonary and hepatic effects of inhaled ozone in rats. Environ Health Perspect. 102 Suppl 10, 61-4. Leikauf, G. D., et al., 1995. Airway epithelial cell responses to ozone injury. Environ Health Perspect. 103 Suppl 2, 91-5. Li, P., et al., 2013. Observational studies and a statistical early warning of surface ozone pollution in Tangshan, the largest heavy industry city of North China. Int J Environ Res Public Health. 10, 1048-61. Lobasso, S., et al., 2010. Lipidomic analysis of porcine olfactory epithelial membranes and cilia. Lipids. 45, 593-602. Malnic, B., et al., 1999. Combinatorial receptor codes for odors. Cell. 96, 713-23. Mann, W. J., et al., 2002. Exposure to 200 ppm of methanol increases the concentrations of interleukin-1beta and interleukin-8 in nasal secretions of healthy volunteers. Ann Otol Rhinol Laryngol. 111, 633-638. Martínez, S. B., et al., 2011. Use of ozone in wastewater treatment to produce water suitable for irrigation. Water Resour Manag. 25, 2109-2124. Medinsky, M. A., Bond, J. A., 2001. Sites and mechanisms for uptake of gases and vapors in the respiratory tract. Toxicology. 160, 165-72. Miller, L. E., Stewart, M. E., 2011. The blind leading the blind: use and misuse of blinding in randomized controlled trials. Contemp Clin Trials. 32, 240-3. Mokoena, M. L., et al., 2011. Appraisal of ozone as biologically active molecule and experimental tool in biomedical sciences. Med Chem Res. 20, 1687-1695. Mokoena, M. L., et al., 2010. Ozone modulates the effects of imipramine on immobility in the forced swim test, and nonspecific parameters of hippocampal oxidative stress in the rat. Metab Brain Dis. 25, 125-33. Mosimann, B. L., et al., 1993. Substance P, calcitonin gene-related peptide, and vasoactive intestinal peptide increase in nasal secretions after allergen challenge in atopic patients. J Allergy Clin Immunol. 92, 95-104.

24

Müller, M., Cotinine. In: J. Angerer, (Ed.), Deutsche Forschungsgemeinsschaft. Essential biomonitoring methods. Wiley-VCH, Weinheim, 2006, pp. 243-253. http://onlinelibrary.wiley.com/doi/10.1002/3527600418.bi48656e0008/full, assessed at 20-April-2018. Muttray, A., et al., 2013. Acute effects of an exposure to 100 ppm 1-methoxypropanol-2 on the upper airways of human subjects. Toxicol Lett. 220, 187-192. Muttray, A., et al., 1999. The exposure of healthy volunteers to 200 ppm 1,1,1-trichloroethane increases the concentration of proinflammatory cytokines in nasal secretions. Int Arch Occup Environ Health. 72, 485-488. Muttray, A., et al., 2004. Acute effects of 1,1,1-trichloroethane on human olfactory functioning. Am J Rhinol. 18, 113-117. Nasr, A. N., 1971. Ozone poisoning in man: clinical manifestations and differential diagnosis - a review. Clin Toxicol. 4, 461-6. Nikasinovic, L., et al., 2003. Nasal epithelial and inflammatory response to ozone exposure: a review of laboratory-based studies published since 1985. J Toxicol Environ Health B Crit Rev. 6, 521-68. Omer, A. R., Walker, P. M., 2011. Treatment of swine slurry by an ozone treatment system to reduce odor. J Environ Protect. 2, 867-872. Ottaviano, G., et al., 2012. Nasal dysfunction induced by chlorinate water in competitive swimmers. Rhinology. 50, 294-8. Paoletti, E., et al., 2014. Ozone levels in European and USA cities are increasing more than at rural sites, while peak values are decreasing. Environ Pollut. 192, 295-9. Parga, J. R., et al., 2003. Destruction of cyanide waste solutions using chlorine dioxide, ozone and titania sol. Waste Manag. 23, 183-91. Pierce, J. D., Jr., et al., 1996. The role of perceptual and structural similarity in cross-adaptation. Chem Senses. 21, 223-37. Prah, J. D., Benignus, V. A., 1979. Decrements in olfactory sensitivity due to ozone exposure. Percept Mot Skills. 48, 317-8. Prueitt, R. L., Goodman, J. E., 2016. Evaluation of neural reflex activation as a mode of action for the acute respiratory effects of ozone. Inhal Toxicol. 28, 484-99. Pryor, W. A., 1992. How far does ozone penetrate into the pulmonary air/tissue boundary before it reacts? Free Radic Biol Med. 12, 83-8. Rawson, N. E., 1999. Cell and molecular biology of olfaction. Quintessence Int. 30, 335-41.

25

Rees, M. D., et al., 2008. Oxidative damage to extracellular matrix and its role in human pathologies. Free Radic Biol Med. 44, 1973-2001. Rice, R. G., Health and safety aspects of ozone processing. In: C. O’Donnell, et al., Eds.), Ozone in Food Processing. Wiley-Blackwell, Chichester, 2012, pp. 265-286. Ross, K., et al., 2012. The impact of the Clean Air Act. J Pediatr. 161, 781-6. Sanderson, W. T., et al., 1999. Ozone-induced respiratory illness during the repair of a portland cement kiln. Scand J Work Environ Health. 25, 227-32. Scannell, C., et al., 1996. Greater ozone-induced inflammatory responses in subjects with asthma. Am J Respir Crit Care Med. 154, 24-9. Schierhorn, K., et al., 2002. Ozone-induced release of neuropeptides from human nasal mucosa cells. Int Arch Allergy Immunol. 129, 145-51. Seeber, A., et al., 2002. Psychological reactions related to chemosensory irritation. Int Arch Occup Environ Health. 75, 314-25. Shaughnessy, R. J., et al., 1994. Effectiveness of portable indoor air cleaners: sensory testing results. Indoor Air. 4, 179-188. Silva Teixeira, C. S., et al., 2016. Unravelling the olfactory sense: from the gene to odor perception. Chem Senses. 41, 105-121. South Coast Air Quality Management District, The Southland's war on smog: fifty years of progress toward clean air. 1997. http://www.aqmd.gov/home/research/publications/50-years-of-progress, assessed at 20-April2018. Todrank, J., et al., 1991. The effects of adaptation on the perception of similar and dissimilar odors. Chem Senses. 16, 467-82. Tuomi, T., et al., 2000. Emission of ozone and organic volatiles from a selection of laser printers and photocopiers. Appl Occup Environ Hyg. 15, 629-34. Turner, M. C., et al., 2016. Long-term ozone exposure and mortality in a large prospective study. Am J Respir Crit Care Med. 193, 1134-42. van Thriel, C., et al., 2003. Physiological and psychological approaches to chemosensory effects of solvents. Toxicol Lett. 140-141, 261-71. Vrablik, K. A., Green-McKenzie, J., 2016. What is a potential source of ozone in the health care environment? J Occup Environ Med. 58, e54-e55.

26

Wu, J., et al., 2018. Olfactory and middle meatal cytokine levels correlate with olfactory function in chronic rhinosinusitis. Laryngoscope. Epub ahead of print, doi 10.1002/lary.27112. Yan, Z., et al., 2016. Inflammatory cell signaling following exposures to particulate matter and ozone. Biochim Biophys Acta. 1860, 2826-34. Zhao, H., et al., 1998. Functional expression of a mammalian odorant receptor. Science. 279, 237-42.

Table 1 Concentrations of environmental pollutants at the measuring station “Mainz-Zitadelle” before the experiments on the different days Group

NO [µg/m³]

NO2 [µg/m³]

PM2.5 [µg/m³]

PM10 [µg/m³]

SO2 [µg/m³]

Ozone (n = 15)

63.5 ± 75.9

60.5 ± 24.7

15.3 ± 8.6

24.9 ± 15.1

6.8 ± 5.4

Sham (n = 13)

63.8 ± 63.1

63.3 ± 28.2

17.6 ± 11.6

26.8 ± 17.0

8.1 ± 6.1

Values are 1-h mean ± SD.

Table 2 Average annual emissions at measuring stations representative for the subjects’ primary residencies O3 [µg/m³]

NO2 [µg/m³]

PM10 [µg/m³]

Group

Mean (Range)

Mean (Range)

Mean (Range)

Ozone (n = 15)

39.9 (35.8 – 43.9)

28.2 (10.1 – 50.5)

20.7 (13.6 – 33.2)

Sham (n = 13)

38.5 (23.1 – 43.9)

33.3 (27.6 – 42.4)

21.3 (18.3 – 24.1)

27

Table 3 Median scores of symptoms of nose and cough

Ozone

Filtered air

Point in time

1

2

3

4

1

2

3

4

Irritation to the nose

0

0

0

0

0

0

0

0

Itching nose

0

0

0

0

0

0

0

0

Dry nose

0

0

0

0

0

0

0

0

Running nose

0

0

0

0

1

0

0

0

Burning nose

0

0

0

0

0

0

0

0

Sensation of bad smell

0

1**

0

1*

0

0

0

0

Cough

0

0

0

2***

0

0

0

0

Items from the extended version of SPES. Points in time: (1) before exposure, (2) shortly after entering the exposure chamber, (3) after one hour exposure, (4) at the end of exposure. p-values < 0.1 are indicated. *: p<0.1, **: p<0.05, ***, p<0.001, Mann-Whitney U test, each.

Highlights  An acute exposure to 0.2 ppm ozone impairs odor detection threshold.  The cause is not clear, different modes of action might play at role.  Olfactory threshold seems sensitive to photochemical air pollutants’ effects.

28