An interdisciplinary team of researchers from federal, state, private, and local organizations have been evaluating aerosolized K. brevis red tide brevetoxin exposures and their possible acute and chronic adverse health effects in humans and animals. The study has been approved by the participating institutional review boards. The study location was Siesta Beach (Sarasota, FL), where prolonged Florida red tides lasting months have become almost an annual event. Although the asthmatic cohort has been studied since 2003, the data used in this analysis were from Florida red tide exposure periods in March 2005 and September 2006.
The study participants consisted of an open cohort of asthmatics with the following characteristics: a self-report of physician-diagnosed asthma; ≥ 12 years of age, history of smoking ≤ 10 years; able to walk on the beach continuously for at least 30 min; and at least 6 months residence in the Sarasota area. For our study, participants spent ≥ 1 hr at the beach in areas with ongoing ambient monitoring. During this time, they could return from the beach at any time if they felt symptomatic, and all participants were encouraged to use any personal medications as needed before, during, and after the study period. Participants were asked not to change their daily asthma management regime on the day of the study.
After obtaining informed consent, we collected detailed baseline information for all subjects (including their medical history and possible confounders) in a baseline questionnaire. Each subject participated in at least one evaluation during an active K. brevis bloom (exposure period) and in one evaluation during a period when there was not a bloom (nonexposure period), although only the exposure period data were used for the analysis. Both evaluations included prebeach and postbeach questionnaires, nasal swab sampling, and spirometry. The prebeach and postbeach questionnaires collected information on recent medical history and medication use, as well as symptoms and possible confounders (e.g., smoking).
Each study participant carried an IOM personal air monitor (SKC Inc., Eighty Four, PA) during their 1 hr of beach exposure. Based on the results of ambient monitoring, the personal monitoring for brevetoxins is important, as the aerosols can vary widely within a stretch of beach, due to wind gusts and direction, the patchy nature of K. brevis
blooms, and variation between participants depending on where and when she/he walked on the beach during an active Florida red tide bloom (Cheng et al. 2005a
Pulmonary function testing was performed according to National Institute for Occupational Safety and Health (NIOSH) standards (NIOSH 1997
) using a portable OMI2000 10-L dry rolling seal volume spirometer (Occupational Marketing, Inc., Houston, TX) before and after 1-hr beach exposure. The spirometry values were forced expiratory volume in 1 sec (FEV1
); the average rate of flow during the middle half of a forced vital capacity (FVC) maneuver (FEF25–75
); peak expiratory flow (PEF), and FVC. Only data conforming to the standard guidelines for collection and interpretation of spirometry measurements were accepted, and all study participants had ≥ 3 reproducible spirograms before and after visiting the beach (Fleming et al. 2007
; NIOSH 1997
For ambient monitoring, a portable, self-contained weather station near a high-volume air particle impactor and the ambient air samplers was used to monitor the air temperature, relative humidity, wind speed, and direction, as described previously (Cheng et al. 2005a
; Naar et al. 2002
; Pierce et al. 2003
). Water samples were collected twice daily in 1-L glass bottles from the surf zone adjacent to the high-volume air sampler locations. The water samples were analyzed for K. brevis
cell counts and for brevetoxin concentrations using both the brevetoxin enzyme-linked immunosorbent assay (ELISA) and liquid chromatography-mass spectroscopy (LC-MS) analysis (Fleming et al. 2007
; Naar et al. 2002
; Pierce et al. 2003
Air samples for toxin and particulate size were collected using two types of high-volume samplers (high-volume filter and high-volume air particle impactors equipped to capture aerosol particles by size) and a personal sampler of the subject breathing zone. We measured brevetoxins by the high-volume sampler and the personal sampler. For the high-volume filter samplers, six 4-hr air filter samples and eight 1-hr air filter samples were measured at a sampling flow rate between 1.6 and 2.0 m3/min. The 1-hr personal exposure of each participant was measured using an individual personal air sampler placed near the breathing zone; the sampling flow rate for the personal samplers was 9-L/min (Cheng YS, personal communication). For the personal samples, we used only the brevetoxin ELISA because it had a lower limit of detection (LOD) than LC-MS, which is important given the low flow rate of the personal air sampler and the small size of the filter paper to be analyzed for brevetoxins. The ambient samples could be analyzed by both LC-MS and brevetoxin ELISA because the flow rate of the ambient air sampler was substantially higher and the filter paper was larger.
The brevetoxin ELISA measures any substance (parent toxins and toxins derivatives/ metabolites) containing the brevetoxin type 2 backbone structure. As such, the reported concentration represents the total amount of brevetoxin-like compounds present in the sample and may also include toxins and/or derivatives that have not yet been chemically described. The LC-MS levels reported in this study represent only the sum of the concentration of five specific breve toxins that could be present in a given sample (PbTx-1, PbTx-2, PbTx-3, PbTx-9, and PbTx-3 42-carboxylic acid). During the 2005 and 2006 exposure periods, the LOD for individual brevetoxins in seawater was 0.03 μg/L using LC-MS and 0.6 ng/sample using the brevetoxin ELISA for all brevetoxins. The LOD for the LC-MS analysis of the ambient air samples was 0.01 ng/m3 for individual brevetoxins; the ELISA LOD for both the ambient and personal samples was 0.6 ng/sample for all brevetoxins combined.
Exposure and health assessment
In previous analyses involving the asthmatic cohort, “exposure” was defined as spending 1 hr on the beach during a study day when a
) K. brevis
cells above background levels (i.e., > 5,000 cells/L) and brevetoxins were detected by LC-MS and ELISA in the water, and b
) brevetoxins were detected in the ambient air monitors by LC-MS and ELISA (Fleming et al. 2005b
; Milian et al. 2007
). However, no attempt was made previously to assign individual exposure levels or to use the personal air sampler data. In this analysis, each subject who had participated in a study during an active Florida red tide was assigned a
) the ELISA brevetoxin level from their individual personal air sampler (personal ELISA); b
) the ELISA brevetoxin level from the hourly ambient air sampler (ambient ELISA) that corresponded to their individual beach walk time; and c
) the LC-MS brevetoxin level from the hourly ambient air sampler (ambient LC-MS) that corresponded to their individual beach walk time. The ambient sampler brevetoxin concentrations were calculated from the hourly samples corresponding to the sampling time of each personal sample. When a personal sample was taken during a portion of two hourly ambient samples, the corresponding ambient concentration was calculated by multiplying the hourly sample with the fraction of time the personal sample was taken during each of the particular hourly samples and then summing the two concentrations.
We asked questions about the presence of symptoms consistent with asthma (i.e., eye, nose, and throat irritation, cough, wheeze, chest tightness, and shortness of breath) before and after spending 1 hr on the beach. If the participant reported any symptom, they were asked if they experienced it as mild, moderate, or severe. We analyzed the symptom data in two ways. First, the number of persons not reporting a symptom before going on the beach but reporting the particular symptom after exposure was counted as being symptomatic from exposure (Fleming et al. 2005b
). Second, for those individuals reporting any respiratory symptom, we devised a respiratory symptom intensity score based on the sum of all respiratory symptoms with their individual symptom intensity of response [i.e., mild (1), moderate (2), and severe (3)] (Milian et al. 2007
); the mean difference in the prebeach walk and postbeach walk symptom scores was then evaluated for each individual.
As with the symptoms, each participant served as their own spirometry control. We present the mean difference of the individual prebeach walk minus postbeach walk spirometry values. A positive mean difference indicated a decrease in lung function after the walk compared with before the walk. Because brevetoxins were measured ≥ 1 mile inshore from the coast during active K. brevis
bloom aerosols when there were strong-enough onshore winds, “coastal residence” was defined as residence on a barrier island or along Sarasota Bay within 1 mile of a coast (Kirkpatrick et al. 2006
). As in previous studies, the use of asthma medications within 12 hr before going to the beach was used as a surrogate for increased asthma severity (Fleming et al. 2007
; Milian et al. 2007
We created the study database in Microsoft ACCESS (Microsoft Corporation, Redmond, WA), with direct data entry during participant interviews. Descriptive and other statistical analyses were performed using SAS statistical software, version 9.1 (SAS Institute Inc., Cary, NC). For all subjects who participated in both March 2005 and September 2006, we used the data with the highest LC-MS ambient measure.
For the symptom data, analyses were performed using the distribution of the exposure measures by quartiles and by above/below the median of the ambient brevetoxin level. For pulmonary function data, we compared the mean differences between the different groups above and below the mean, the median, and by quartiles, depending on the particular data distribution. Statistical hypothesis testing was performed using paired t
-test for continuous data and McNemar’s test for categorical data to compare pre beach and postbeach data (Kleinbaum et al. 1982
). The McNemar’s test at 0.05 level of significance uses only the data on the diagonal of the two-by-two table, which indicates a change; thus, subjects who came to the beach reporting no symptom and left reporting a particular symptom were compared with subjects who came to the beach reporting a symptom and left reporting no particular symptom. For the symptom score, the pre beach walk value of each individual was compared with that individual’s post-beach walk value using a two-tailed paired t
-test at the 0.05 level of significance. In addition, we performed correlation analyses between individual pulmonary function tests (PFTs) and the ambient measures, as well as multi variable regression analysis with differences between pre- and post-PFTs (as the outcome variable), ambient levels, medication use, sex, age, and geographic proximity of residence to the coast.