Exposure to a Fungal Volatile Compound: Significance of Effects

Referencing: Acute Effects of a Fungal Volatile Compound

In contrast to what Wålinder et al. (2005) concluded in their article "Acute Effects of a Fungal Volatile Compound," I interpret the article to report essentially no effects beyond chance. In all, the authors carried out some 76 comparisons (each one representing a time point and an exposure vs. control measurement) if you take blink frequency as a single comparison. The authors reported finding 5 "significant differences" out of 76 comparisons. Of the reported significant differences, one (blink frequency) is misleading, as discussed below. Of the remaining 75 comparisons, 4 differences at a p-value of < 0.05 might be expected by random chance. This is without applying Bonferroni's adjustment for multiple comparisons; using this adjustment, a p-value of approximately < 0.0007 would be required for a single comparison to be statistically significant. None of the differences reported reached this level.

Wålinder et al. (2005) reported that the subjects showed increased "blink frequency" during 3-methylfuran (3-MF) exposure (Table 1), but the frequency was higher in the exposure phase at time 0, about 9 for 3-MF exposure versus 6.5 for the control air phase. From the data in Table 1, it appears that both groups had lower overall blinks per minute compared with baseline during the trial (Figure 2). Reporting blinking was higher during exposure, and not noting that it was higher at baseline is disingenuous.

Wålinder et al. (2005) may have mislabeled tear break-up, but as it reads in the legend for Table 1, a negative value indicates a decrease; therefore, the 6 sec given for measured break-up time after 3-MF exposure (Table 1) indicates that it was increased (i.e., longer to tear break-up), which is better. Is this correct? Also, was the observer who measured the tear break-up blinded to the exposure?

Finally, of the four lung measurements taken, the only comparison with a p-value of < 0.05 was the small 100 mL change for forced vital capacity (FVC) right after exposure (Table 4). How do the authors interpret this change in FVC in view of the fact that there was no significant change in forced expiratory volume in 1 sec (FEV₁)?

The author has provided expert testimony in mold litigation.

Andrew Saxon
Department of Medicine
UCLA Medical School
Los Angeles, California
E-mail: asaxon@mednet.ucla.edu

Reference

---

Exposure to a Fungal Volatile Compound: Wålinder et al. Respond

There are different opinions on the use of Bonferroni's corrections. Everitt (1995) stated that it gives too conservative estimates if there are more than five tests performed. In environmental medicine, one exposure can have different health effects, so it is reasonable to test for different types of effects on different organs. We prefer to perform conventional statistical tests, without Bonferroni correction, and look at the pattern of significant effects and their biologic plausibility.

We did not perform 76 comparisons (Wålinder et al. 2005); we actually performed 25 tests on 13 physiologic variables based on differences before and after exposure. A fourteenth variable (vital staining) was tested only after exposure. Repeated measurement analysis was performed on blink frequency (60 consecutive measurements of 2 min each) and one questionnaire with 10 questions was administered at six different times. This is a total of 37 tests performed on 25 variables, having five significant values, of which one was highly significant (p < 0.001).

Moreover, all tests point in the same direction--mucosal effects of the exposure. We did not find the same effects over time for control exposure. There is, of course, the possibility that some of the significant effects were due to chance, and we were quite modest in our conclusions, using the words "may" or "might be." Because our study is the first exposure-chamber study on 3-methylfuran (3-MF), more studies are needed to determine final conclusions.

Blink frequency was only measured during the 2 hr of exposure in the chamber. Therefore, there is no preexposure baseline data available at time 0. Eye effects occur quickly; a rapid effect of the exposure on blinking frequency can occur during the first 2 min of exposure to 3-MF inside the chamber, possibly followed by later adaptation (8.8 blinks/min during the first 2 min, compared with a mean of 7.6 blinks/min during the whole period of exposure).

It is true that there was a numerical increase in break-up time at exposure, which could be in agreement with increased blink frequency. The fatty layer on the tear film is produced by the glands of the eyelids. Therefore, an increased blinking frequency could produce more secretion from the meibomian glands and therefore a longer break-up time.

Regarding lung function, transient effects of environmental exposure (as well as diurnal variation, which we controlled for by performing the experiment at the same time) may affect either forced vital capacity (FVC) or forced expiratory volume in 1 sec (FEV₁), or both. Physiologically and numerically, the decrease was of the same order, but statistically the outcome was different. The decreases were 0.1 L for FVC and 0.08 L for FEV₁ after exposure to 3-MF. The magnitude of the effect was clinically small, but it was significant at group level for FVC. Small pulmonary effects may have large health effects in a population (Künzli et al. 2000.)

The authors declare they have no competing financial interests.

Robert Wålinder
Dan Norbäck
Gunilla Wieslander
Department of Medical Sciences/Occupational and Environmental Medicine
University Hospital
Uppsala, Sweden
E-mail: robert.walinder@medsci.uu.se

Lena Ernstgård
Gunnar Johanson
Division of Work Environment Toxicology
Institute of Environmental Medicine
Karolinska Institutet
Stockholm, Sweden

References