Mar. Drugs 2008 , 6 , 389-406; DOI: 10.3390/md20080018 

# Marine Drugs
ISSN 1660-3397 www.mdpi.org/marinedrugs A rticle OPEN ACCESS



## Recreational Exposure to Low Concentrations of Microcystins During an Algal Bloom in a Small Lake

Lorraine C. Backer 1,*, Wayne Carmichael 2 , Barbara Kirkpatrick 3 , Christopher Williams 4 , Mitch Irvin 5 , Yue Zhou 5 , Trisha B. Johnson 6,7 , Kate Nierenberg 3 , Vincent R. Hill 6 , Stephanie M. Kieszak 1 and Yung-Sung Cheng 5

1 National Center for Environmental Health, Ce nters for Disease Control and Prevention, 4770 Buford Highway NE, Chamblee, Georgia 30341 , USA 2 Department of Biological Sc iences, Wright State Universit y, 3640 Colonel Glen Highway, Dayton, Ohio 45435. Current address: 42184 Tw eedle Lane, Seaside, Oregon 97138 , USA 3 Mote Marine Laboratory, 1600 Ken Thompson Parkway, Sarasota, Florida 34236 , USA 4 Greenwater Laboratories, 205 Zaeg ler Drive, Pakatka, Florida 32177 , USA 5 Lovelace Respiratory Research Institute, Inhalation Toxicology Laboratory, P.O. Box 5890, Albuquerque, New Mexico 87285 , USA 6 National Center for Zoonotic, Vector-Borne, and Enteric Diseases, Centers for Disease Control and Prevention, 4770 Buford Highway NE, Chamblee, Georgia 30341 , USA
7 Atlanta Research and Education Foundati on, 1670 Clairmont Road, Decatur, Georgia 30033 , USA
* Author to whom correspondence should be ad dressed; E-mail: lbacker@cdc.gov; Tel: +1-770-488- 3426; Fax: +1-770-488-3450.
Received: 6 February 2008; in revised form: 5 Ju ne 2008 / Accepted: 19 June 2008 / Published: 26 June 2008

Abstract: We measured microcystins in blood from people at risk for swallowing water or inhaling spray while swimming, water skiing, je t skiing, or boating during an algal bloom. We monitored water samples from a small lake as a Microcystis aeruginosa bloom developed. We recruited 97 people planning recr eational activities in that lake and seven others who volunteered to recreate in a n earby bloom-free lake. We conducted our field study within a week of finding a 10-μg/L microcystin concentr ation. We analyzed water, air, and human blood samples for water quality, potential human pathogens, algal taxonomy, and microcystin concentrations. We interviewed study participants for demographic and current health symptom in formation. Water samples were assayed for potential respiratory viruses (adenoviruses a nd enteroviruses), but none were detected. We did find low concentrations of Escherichia coli , indicating fecal contamination. We foundMar. Drugs 2008, 6 390

low levels of microcystins (2 μg/L to 5 μg/L) in the water and (<0.1 ng/m 3 ) in the aerosol samples. Blood levels of microcystins for all participants were below the limit of detection (0.147μg/L). Given this low exposure level, study participants reported no symptom increases following recreational exposure to mi crocystins. This is th e first study to report that water-based recreational activities can ex pose people to very low concentrations of aerosol-borne microcystins; we recently conduc ted another field study to assess exposures to higher concentrations of these algal toxins.

Keywords: Aerosol exposures, blue-green algae, cyanobacteria, microcystins, Microcystis aeruginosa, waterborne exposures

Introduction
For hundreds of years, harmful algal blooms (HABs) and HAB-related toxins have been associated with incidents of wild and domestic animal poisonings and outbreaks of human illnesses [1, 2, 3, 4]. In documented incidents, illnesses were typically associated with eating seafood contaminated with marine HAB toxins, drinking water contaminated w ith cyanobacterial (blue-green algal) HAB toxins, or with failures to protect water used for medi cal purposes, such as ki dney dialysis [5, 6]. In addition to consuming toxins directly in contam inated food or water, people are, during outdoor recreational or occupational activities, exposed to HAB-related toxins in their environment. For example, recent work has shown that brevetoxins produced by Karenia brevis , the organism responsible for Florida red tides, can be incorporated into mari ne aerosols and blown onshore [7]. When these aerosols are inhaled, th ey induce respiratory symptoms in beach visitors and in healthy lifeguards [8, 9] and detectable changes in pul monary function in peopl e with asthma [10, 11]. Stewart et al. [12] reviewed case reports a nd anecdotal references that de scribe illnesses associated with exposure to cyanobacteria and related toxins during recrea tional activities in freshwater environments. The reported symptoms included hay-fe ver-like symptoms, gastroenteritis, pyritic skin rashes, and allergic reactions. Othe rs, however, reported more severe acute reactions in cluding severe headaches, fever, and blistering in the mout h. One anecdotal report (John Burns, personal communication, 2001) suggested that people exposed to aerosols on golf courses and lawns watered with local pond water experience re spiratory irritation. In addition to these case reports and anecdotes, epidemiologic studies examining recreational exposure s to cyanobacteria have been published [13, 14, 15, 16, 17]. Most studies did not find an association between recreational e xposure to cyanobacteria and adverse health effects. However, Pilotto et al. [17] did find a significant trend of increasing symptom reports with longer water contact time and exposure to higher cyanobacteria counts. In addition, people in this study who were exposed for more than 1 hour to water with low concentrations of cyanobacteria (> 5,000 cells per ml) were more likely to complain of at least one symptom during the 7 days following exposure than were people w ho were exposed to waters that did not contain cyanobacteria (odds ratio = 3.44; 95% c onfidence interval (CI), 1.09 – 10.82). More recently, Stewart et al. [18] conducted a study of over 3,500 people who used personal water craft on small freshwater lakes in southeast Queensland (Australia ), coastal New South Wales, orMar. Drugs 2008, 6 391

Florida (US). In the follow-up phone interview conduc ted at least 3 days following the water-contact activities, people who used personal watercraft on lakes with high levels of cyanobact eria (cell surface area >12.0 mm 2 /mL) were 2.1 (CI, 1.1 – 4.0) times more likely to report symptoms—particularly respiratory symptoms—than were pe ople who used their personal waterc raft on lakes with low levels of cyanobacteria (ce ll surface area < 2.4 mm 2 /mL). Using available evidence that s upports an association between e xposure to waters containing high concentrations of cyanobacteria and subsequent a dverse health effects, many entities, including the World Health Organization (WHO), Australia, and some other European countries, have developed guidelines for managing recreational waters w ith cyanobacterial bloom s [19, 20]. The WHO guidelines, for example, are based on the following three tiers of potential public health threat: 1) low probability of adverse health effects from water with ≤ 20,000 cells/mL or 10 μg chlorophyll-a/L where cyanobacteria predominate, 2) moderate probabi lity of adverse health effects from waters with 100,000 cells/mL or 50 μg chlorophyll-a/L, and 3) high probability of adverse health effects from contact with, ingestion, or inhalation of cyanobacteria when alga l scum appears on the water surface. These guidelines are based on cell concentrations ra ther than on cyanobacteria toxin concentrations. Because a given bloom may or not produce toxins, it is possible that the guidelines may be too conservative in terms of limiti ng access to recreational waters. Ep idemiologic studies to assess symptoms and to measure concentrations of cya nobacterial toxins in the environment (water and aerosols) could provide data n eeded to develop more specific recreational exposure guidelines. Our primary objective was to assess whether we could measure microcystins in blood from people at risk for swallowing water or inhaling spray while engaged in water-related recreational activities (e.g., water skiing, tubing, swimming, and using personal watercra ft) in a lake during a microcystinproducing algal bloom. In additi on, we were interested in de termining whether microcystin concentrations in aerosol and water samples were a ssociated with microcystin concentrations in blood and with self-reported symptoms.

Results and discussion
Study site

We were aware of a number of sma ll recreational lakes in Michigan, New York, Ohio, and other states that historically experienced microcystin-producing algal blooms in late su mmer, and we corresponded with colleagues conducting routine monitoring of these lakes. Base d on previous experience with accidental exposures to water contaminated with micr ocystins, we expected that people involved in activities that involve ingesting water (i.e., swimming) or inhaling aerosols (i .e., jet-skiing, water skiing, or sailing a small boat) should receive enough exposure to allow us to detect microcystins in their blood. The minimum amount of microcystins de tectable in blood was a bout 0. 9 ng microcystins in 10 ml of blood (approximately 0.1 ppb). This was very close to the 1 ng limit of detection for the ELISA assay. Accordingly, we planned to conduct the field st udy within a week of receiving monitoring results that the microcystin concentrations in one of the lakes was at least 10 μg/L. Delays in conducting the tests and unpredictable change s in local weather can, however , rapidly change algal bloom characteristics. For this field study, water sample analyses completed after the study found that evenMar. Drugs 2008, 6 392

with a significant, visible bloom of organisms capable of producing microcystins, the concentrations of microcystins were actually in the range of 2–5 ug/ L. Thus people may have been exposed, but the waterborne microcystin concentrations were not high enough to detect micr ocystin in blood samples from our study subjects. Nevertheless, in this field study we verified that inha lation is a possible route of exposure to microcystins, and thes e data can be compared with data from studies that used the same protocol in lakes with potentially higher exposures.
Water Samples
We collected 24 water samples (four sample site s, each sampled in the morning and afternoon, on each of the 3 study days, August 4-6, 2006) for analys is of water quality, alga taxa, and microcystin concentrations from the lake with the algal bloom. Table 1 contai ns the ranges of water quality parameters tested over the 3-day study period as well as weather da ta collected during that same period. In general, cyanobacterial popul ations are found and thrive in warm, calm waters with high concentrations of nutrients and characterized by consistent, rela tively high pH and dissolved oxygen (DO) levels. High-pH (8–10) and DO (above saturation) would be a result of relatively high population densities and are not a prer equisite for growth. Still, if potenti ally toxigenic species were present, warm temperatures (25–30 o C), low wind action (0–3 mph), and high nutrient loads (phosphorus and nitrogen), together with clear sunny skies, would stimulate growth and increase the potential risk for toxin production. Several factors contributed to the weak bloom and low concentrations of microcystins in the water we tested. First, microcystins are endotoxins stored and isolated inside algal cells, thus they do not readily diffuse or leak into th e aquatic envi ronment. Under normal envi ronmental conditions in a healthy, reproductively active toxi n-producing bloom event, 95% to 98% of the toxins would be intracellular. Accordingly, we would expect to fi nd toxin only in water containing cells. Second, during the sampling period for the lake with the bloom, winds generally increased in speed from morning to afternoon, thus disorganizing bloom accumu lations and diluting micr ocystin concentrations by scattering algal cells. Third, Microcystis species are buoyant, surface- floating algae that tend to accumulate on or near shore as winds blow them landward. Three of the f our samples sites where water samples were collected were open water—thus we would not expect hi gh toxin concentrations unless the bloom was extensive. For example, early one morning before the winds had increased in strength, we sampled a small accumulation of cells a nd found microcystin concentrations of 43 ug/L. Later that morning and in the af ternoon we did not observe cell accumulations, and in the water samples, toxin concentrations remained relativel y low (3 ug/L – 5 ug/L). In addition, during the study period a storm with significant winds (> 5 mph) further dispersed the bloom.
Algal taxonomy
We assessed algal taxonomy in 12 samples collected during the morning of each of the 3 study days from the exposure lake at the four sampling stat ions. The range of phytoplankton concentrations was 175,000 cells/mL to 688,000 cells/mL. Over 95% of the cells were cyanobacteria.Mar. Drugs 2008, 6 393

Table 1. Microcystin concentrations (average of am and pm samples), water quality parameters (average of am and pm samples) , and weather parameters measured during the study.

Station 1 Station 2 Station 3 Station 4

Water quality parameter

(units) Day 1 Day 2 Day 3 Day 1 Day 2 Day 3 Day 1 Day 2 Day 3 Day 1 Day 2 Day 3

Microcystin concentratio n (μg/L ) 2 2 3.5 2 4 3.5 2 2.5 2 2.5 2 3

Water temperature (°C) 28.9 27.2 26.8 28.7 27.7 27.1 28.2 27.3 26.5 27.7 27.9 27.2

pH 8.9 9.0 8.9 8.9 8.8 8.8 8.8 8.5 8.5 9.0 9.0 9.0

Dissolved oxygen (mg/L) 8.7 9.2 9.0 8.8 8.2 7.8 9.7 8.1 7.2 10.2 9.72 9.9

Salinity (ppt) 0.17 0.18 0.18 0.17 0.18 0.18 0.17 0.17 0.18 0.18 0.18 0.18

Turbidity (NTU) 12.4 18.0 0.1 16.0 17.4 3.2 22.4 11.2 0.2 28.4 1.6 21.3*

Sample site depth (m) 2.5 2.4 2.3 2.5 2.8 2.8 1.2 1.2 1.3 0.4 0.4 0.4

Chlorophyll (μg/L ) 14.0 15.2 18.2 16.2 16.4 20.2 15.3 17.4 19.5 19.2 17.3 16.8

Total dissolved solids (mg/L) 0.249 5 0.252 9 0.248 2 0.250 0 0.244 8 0.247 4 0.246 2 0.243 8 0.246 6 0.247 9 0.243 2 0.245 4

Weather

Air temperature (°C) 28.8 28.4 27.8 29.2 29.4 28.0 29.3 29.2 24.7 30.0 28.2 25.7

Cloud cover (%) 7.5 55 100 0 60 95 2.5 80 90 10 88 65

Wind speed (mph) 3.6 6.4 2.0 5.2 5.2 6.8 4.0 3.0 8.2 6.0 2.2 6.0

Wind direction (0 - 360°) 315 172 232 175 160 210 298 150 225 180 200 248

* Only the afternoon measurement was available.Mar. Drugs2008,6394Table 2.Densities of potentially toxigenic cyanobacteria in water samplesfrom four sampling stations on each of 3 days in the study lakewith the algal bloom.Potentially Toxigenic Cyanobacteria(PTOX) (cells/mL)Station 1Station 2Station 3Station 4GeneraSpeciesDay 1Day 2Day 3Day 1Day 2Day 3Day 1Day 2Day 3Day 1Day 2Day 3Anabaena Macrospore4,925 5,746 11,492 1,437 10,056 7,183 1,437 456 8,619 7,183 2,873 17,239Spiroides2,062 3,932 983 983 2,949 2,873 3,932 286 2,949 3,392 2,949 7,863Sp.5,444 6,351 12,702 3,969 11,432 3,811 6,351 2,722 1,684 3,176 7,621 6,447Aphanizomenon Aphanizomenonoides/gracile25,923 24,295 18,146 24,195 25,404 39,921 18,146 2,592 25,518 25,404 25,404 30,243Issatschenkoi7,777 3.024 3,024 2,268 3,629 2,268 3,024 756 2,268 3,629 3,629 3,024Cf. flos-aquae5,444 3,176 9,527 6,351 7,621 11,432 3,176 2,722 3,176 7,621 7,621 6,351Cylindrospermopsis Raciboskii0 0 1,361 340 0 3,266 0 1,167 1,276 0 0 6,850Microcystis Cf. botrys20,220 5,897 15,726 5,897 18,872 37,744 7,863 6,740 22,115 7,863 7,863 29,487Wesenbergii3,213 1,870 3,213 12,853 2,210 6,427 1,190 85 3,213 1,530 1,530 6,427Sp.6,049 240 6,049 240 80 29,033 80 480 51,414 0 3,024 9,073Total PTOX cells/mL81,057 54,431 82,223 58,533 82,253 143,958 45,199 18,006 122,232 61,517 62,514 122,959Mar. Drugs 2008, 6 395

Table 2 contains the densities of potentially toxigenic cya nobacteria (PTOX-C). During the sampling period, densities ranged from 54,000 total cells/mL to 144,000 total cells/mL. The dominant genera of PTOX-C reported in water samples were Anabaena , Aphanizomenon , Cylindrospermopsis , and Microcystis . The two documented microcystin-p roducing genera present were Anabaena and Microcystis . No Planktothrix species were observed. The dominant species present were Anabaena
macrospora , Aphanizomenon aphanizomenoides/gracile , and Microcystis cf. botrys

. Aphanizomenon aphanizomenonoides/gracile is, however, a suspected microcystin producer. Because the toxin levels were very low, we could not examine in the collect ed water samples any poten tial relationship between the number of microcystin-producing PTOX-C a nd the concentrations of microcystins.
Air Samples
On each of the three study days we detected microcystins at levels near the level of detection (LOD) (0.0037 ng/m 3 ) on each stage of the high volume, airborne particle impactor. The flow rate, sampling time, and total concentration of microcystins from th e five stages and the back-up filters, respectively, were as follows: day 1, 1.17 m 3 /min, 360 min, 0.050 ng/m 3 ; day 2, 1.15 m 3 /min, 386 min, 23 ng/m 3 ; and day 3, 1.22 m 3 /min, 484 min, 0.057 ng/m 3

. We deployed 10 personal air samplers on study da ys 1 and 2, and 4 personal air samplers on study day 3. A summary of the mass and air concentration of microcystins as determined on each study day using the personal samplers is presented in Table 3. We were able to detect microcystins in 9 of 10 samples collected on day 1 and 6 of 9 samples on day 2. On day 3 we did not collect measurable concentrations of microcystin s using personal air samplers.

Table 3. Mass and air concentrations of microc ystins collected on each study day using personal samplers carried by study participants or pl aced on their boats.

Study Day Mean Sampling Time (min) Mass of microcystins on filter (ng) Air concentration of microcystins (ng/m 3 )

Mean Standard Deviation Range Mean Standard Deviation Range

1 88.9 0.07 0.07 0.006-0.223 0.08 0.09 0.005-0.319
2 134 0.06 0.09 0.017-0.29 0.07 0.014 0.020-0.456
3 NA <LOD NA <LOD <LOD NA <LOD Note: the air pump fl ow rate was 0.0106 m 3 /min.
Waterborne pathogens

Stewart et al. [18] found an increase in re spiratory symptoms in people exposed to higher levels of cyanobacteria (surface area > 12.0 mm 2 /mL) than in people exposed to lower levels of cyanobacteria (surface area < 2.4 mm 2 /mL). It is possible that respiratory sy mptoms could be caused by infections from viruses rather than cyanobact eria. Thus, for our study, we added the analysis of the presence of adenoviruses, as well as entero viruses in the lake water.Mar. Drugs 2008, 6 396

Each 10-L water sample was tested for adenoviru ses using real-time PCR and enteroviruses using real-time reverse-transcription (R T)-PCR. Adenoviruses and enteroviru ses were not detected in any water sample. Based on published detection limits for these assays, the testing protocol used in this study resulted in detection limits of approximately 1,250 adenovirus gene equivalent copies (GEC) per 10-L water sample and 200 enterovirus plaque-formi ng units (PFU) per 10-L sample. Thus, these data indicate that adenoviruses and enter oviruses were not present at substa ntial concentrati ons in the lake with the Microcystis bloom. In some samples, however, PCR inhibition was observed based on the delays in CT values measured for water samples seed ed with an inhibitor sta ndard versus CT values for seeded reagent-grade water controls. The range of CT value differences between seeded samples and controls was 0.6 to 9.3, with an average difference of 2.8 ± 2.0 CT values. This inhibition was likely due to the presence of bloom-associated cons tituents in the water samples—such as algal matter and other organic material (inclu ding humic and fulvic acids)—that are generally reflected by the relatively high turbidity da ta reported in Table 1. The positive real-time PCR tests for Escherichia coli (data not shown), indicated that the organism was present at low levels (10 colony-forming units [cfu]/mL to 100 cfu/100 mL) in water from the lake with the bloom. E. coli is an established, fecal-specific water qua lity indicator that has been shown to correlate with reports of gastroin testinal illness in people exposed to the water du ring recreational activities [21, 22]. Thus despite the absence of any fecal-associated virus cont amination in the lake with the algal bloom, we did find evidence of fecal contamination (i.e., the presence of E. coli ).
Study participants
We recruited 104 study participants from lake vis itors planning recreationa l activities that would generate aerosols, such as boating and using personal watercraft. Ninety -seven participants planned to use the lake with the bloom (the exposed group) and 7 planned to use a nearby lake with no bloom (the unexposed group). We obtained complete questionnaire data from 96 people in the exposed group and 7 in the unexposed group. For the exposed group, the age range was 12 years to 67 years. Nine ty-three (97%) were white, and 4 (4%) were of another race—no Hispanics were in the study. Forty-five (47%) of the exposed participants were female. Of the 96 exposed participants, fifty-seve n (59%) reported that during the 7 days before the study they had partic ipated in water-relate d recreational activities on the study lake. In addition, these exposed participants reported that in the year befo re the study, 75 (77%) had boated on, 34 (35%) had fished in, and 72 (74%) had swum in the study lake. Two (2%) reported using dietary supplements containing blue-green algae. Study participants in the expos ed group reported the following act ivities during the study period: swimming (73 [76%]), boating (34 [ 51%]), tubing (20 [30%]), riding pe rsonal water craft (13 [14%]), wake-boarding (8 [12%]), wading (3 [4%]), and fishing (2 [3%]). Of the study participants who swam, the amount of time ranged from 5 min to 180 min. Of those who rode personal watercraft, the amount of tim e ranged from 15 min to 120 min. Of those who did the other activit ies, the amount of time ranged from 10 min to 300 min Sixty-one (64%) of the exposed group reporte d that during the study period they had put their head underwater, and 39 (41%) reported they had swallowed water. For the unexposed group, the age range was from 15 years to 58 years. All of the unexposed participants were white and non-Hi spanic, and 4 (57%) were female. Of the 7 unexposed participants, 6 (86%) reported they participated in water-related recreational activities on the study lake (exposedMar. Drugs 2008, 6 397

site) during the 7 days before the study and in the year before the study. No one reported using dietary supplements containing blue-green algae. The seven participants in the unexposed group re ported only swimming dur ing the study period. Six (86%) were in the water for 60 min, and one (14 %) was in the water for 90 min. Six (86%) reported that during the study period they had put their head underwater, a nd 1 (14%) report ed swallowing
water. Table 4 contains a summary of symptoms reported by both expos ed and unexposed participants during the interviews. As a group, st udy participants reported more symp toms in the 7 days before the study than during study or during th e 7 to 10 days after the study. In addition, as a group, study participants tended to report mo re symptoms (e.g., cough, sore throat for the exposed group, skin complaints for the unexposed group) immediately be fore doing study activities than immediately after such activities. A single participant in the contro l group reported five skin-related symptoms before going into the water but not after coming out of the wa ter. No differences appeared in the frequency of reported symptoms between the exposed and cont rol groups during the 7 days before the study, immediately before or immediat ely after doing study activities, or during the follow-up period. We hypothesized that exposure to aerosolized microcystins woul d result in acute dermal and respiratory symptoms; thus, we expected people to report more symptoms immediately after study activities than during the week be fore the study. However, more people reported symptoms before doing study activities than afte r. In addition, par ticipants reported more symp toms for the period of 7 days before the study than either immediately before or after doing study activ ities. Also, more than half of the participants in the exposed group and all but one of the participants in the control group had participated in water-re lated recreational ac tivities on the study lake in the week before the study. Finally, the number of people reporting symptoms during the seven to 10 days after the exposure increased to levels that were more consistent with the number of people reporting symptoms for the period of 7 days before the study than with the number of people reporting symptoms either immediately before or immediatel y after doing study activities. While these differences in symptom reporting are not significantly different, the trend suggests that exposure to aerosolized microcystins may result in symptoms with onset a day or few da ys after the exposure, i.e., during the week after weekend activities on the blooming lake. This possi bility could be addressed in a future study by identifying a study population that uses a lake that historically develops a summer bloom of microcystin-producing cyanobacteria and conducting sy mptom surveys before their first visit to the lake and then periodically throughout the summer.
Blood samples
We collected usable blood samples from 96 e xposed and 6 unexposed participants. Of the 102 blood samples, 101 samples tested below the limit of detection according to the limit of detection described in the Envirologix Kit (0.147 μg/L). Only one blood sample showed detectab le levels of microcystins—about 1.0 μg/L. LC/MS analysis of this sample showed the absence of microcystin-LR, -RR, and -YR. Given the LC/MS resu lts, we concluded that this sample represented either a false positive or exposure from microcystin(s) not identified as microcystin-LR, -RR, or -YR. We did not identify anything unusual about this particular study participant (e.g., they did not use blue-green algae food supplem ents).Mar. Drugs 2008, 6 398

Table 4. Self-reported symptom data from study participants engaged in recreational activities in a lake with an alga l bloom and a lake without a bloom.

Symptom Study participants doing recreational activities in the lake with the algal bloom Study participants doing recreational activities in the lake without an algal bloom

7 days before study Before studyactivities After study activities 7-10 days after study activities 7 days before study Before study activities After study activities 7-10 days after study activities

Respiratory symptoms Sore throat 7 (7) 4 (4) 3 (3) 8 (8) 2 (29) 0 0 1 (14) Congestion 19 (20) 9 (9) 10 (10) 23 (24) 2 (29) 0 0 1 (14) Cough 5 (5) 4 (4) 1 (1) 5 (5) 1 (14) 0 0 1 (14)

Throat irritation 9 (9) 6 (6) 3 (3) 7 (7) 1 (14) 0 0 1 (14)

Eye irritation 2 (2) 1 (1) 1 (1) 4 (4) 0 0 0 1 (14)
Other respiratory complaint 6 (6) 4 (4) 1 (1) 1 (1) 0 0 0 1 (14)

Dermatologic symptoms Itchy skin 10 (10) 6 (6) 8 (8) 11 (12) 1 (14) 1 (14) 0 2 (29) Red skin 7 (7) 6 (6) 2 (2) 2 (2) 2 (29) 1 (14) 0 0 Hives 3 (3) 2 (2) 0 2 (2) 0 0 0 0
Skin irritation 3 (3) 1 (1) 1 (1) 2 (2) 2 (29) 1 (14) 0 0

Rash 5 (5) 4 (4) 2 (2) 5 (5) 2 (29) 1 (14) 0 0
Other skin problem 4 (4) 4 (4) 3 (3) 2 (2) 1 (14) 1 (14) 0 0

Other symptoms Earache 7 (7) 3 (3) 2 (2) 4 (4) 0 0 0 0 Agitation 3 (3) 3 (3) 0 1 (1) 0 0 0 0 Headache 20 (21) 6 (6) 5 (5) 9 (9) 4 (57) 0 0 1 (14)
Abdominal pain 3 (3) 1 (1) 0 2 (2) 0 0 0 1 (14)

Diarrhea 3 (3) 1 (1) 1 (1) 3 (3) 0 0 0 0
Note: The data are from questionnaires administered for the week before st udy activities, immediately before and after doing study activitie s, and during the telephone follo w-up interview. Symptoms listed are those reported by more than tw o study participants in any of the four questionnaires. Values are number of study participants reporting the symptom (%).Mar. Drugs 2008, 6 399

Experimental Section
Study approval
This study was approved by the Ins titutional Review Board of the Centers for Disease Control and Prevention.
Environmental sampling sites
We used four sampling sites located along the major ax is of a lake that had historical water quality data previously collected by Grand Valley Stat e University. Each time sampling was performed latitude and longitude coordinate s for all sample stations were recorded using a geographical positioning system (Magellan Map 330X, Magellan, Santa Clara, CA).
Water and plankton collection
Water samples were collected twice each day (AM and PM) at each sampling station for the identification of the algae presen t. Algal samples were collected at a depth of 0.15 meters. Water samples used for algal identification were immediat ely preserved with 10% L ugols solution and stored under dark, cool conditions. Water samples were collected for microcystin an alyses at a depth of 0.15 meters by directly immersing sample containers in ambient water. Befo re collection all sample bottles were rinsed with ambient water. In general, 4 liters per sample site were collected. To ensure that algae in samples remained alive and at low metabolic states until processing or preservation, collection bottles were stored in coolers under cool condi tions (i.e., ice packs). To maintain algal health, bottles were also properly vented. After collection, all samples were stored chilled and mailed to GreenWater Laboratories/CyanoLab via overnight mail the day after the st udy was completed. For algal identification and micr ocystin analyses, general water quality parameters (e.g., water temperature, dissolved oxygen, pH, specific conductivity, salinity, and tu rbidity) were measured at all sample sites twice per day at the same time as collection of the water samples. General water quality was measured by using a HACH multi-parameter da ta sonde (Hacj Company, Loveland, CO) supplied by Dr. R. Rediske of Grand Valley State Universit y. Water quality parameters were measured at a depth of 0.15 meters. At the time of water collec tion ambient environmental conditions (cloud cover, wind speed, wind direction, and air temperature) we re recorded at each site. Grand Valley State University performed water chemistry on the morning of August 4 th , 2006.
Raw water sample preservation and algal taxonomy analysis
Samples were mixed to distribute evenly the phytoplankton cells. A 100-mL aliquot was removed and poured into a 125-mL amber polyethylene bottl e and preserved with Lugol’s solution (0.05–1.0% by volume depending on cell density). For each sample to be analyzed, a cleaned Utermöhl counting chamber was constructed. Depending on the phytoplankton density of the sample, a settling tower of 5, 10 or 25 mL was used. The tower was secured to the ba se using a thin film of high vacuum grease. The Lugol’s preserved sample was shaken for 20 seconds to evenly distribute phytoplankton cells, and the appropriate volume added to the set tling tower. A cover glass was pla ced on top of the tower, and theMar. Drugs 2008, 6 400

sample was allowed to settle in the dark in a vi bration-free location. Minimum settling times were between 17 hrs for 5-mL samples, 34 hrs for 10-mL samples and 74 hrs for 25-mL samples. After settling was complete, the tower was slid from the base of the counting chamber to remove overlying water, and the chamber was cove red with a second cover glass. Enumeration was performed on a Nikon Eclipse TE200 (Southern Micr oscopes, Inc., Mebane, NC) inverted microscope equipped with phase-contrast op tics. One ocular was fitted with a whipple disc and used to define the area of the fields to be counted. Specimens that went beyond the left and bottom edges of the whipple grid were not counted. Befo re beginning enumeration, the slide was scanned at low power to ensure a relatively uniform cell distribution. Only intact, viable cells were counted. Counts were made as natural units (i.e., cells, filaments, colonies) per a given volume of water. The goal was to count a total of 400–600 cyanobacterial natural units per sample. A minimum of 10 and a maximum of 50 fields were counted at both 400X and 200X. An additional scan of the entire slide was made at 100X to count large or rare taxa, or bot h. A maximum of 100 fields combined at 200X and 400X was performed on a given sample. Data were entered into a spreadsheet that calculated natural units/mL. To calculate cells/mL, the average number of cells for colonies and filaments was determined and the units/mL multiplied by these values.
Analysis of water samples for microcystins concentrations
To break up cells, water samples were subsampled and sonicated, then filtered and stored at 4 o C for toxin analyses. Microcystin anal yses were performed within 24- 48 hours of receipt of samples. Enzyme linked immunosorbent assay (ELISA) was utilized for the determin ation of the concentration of total microcystins present. The ELISA assay is based on the polyclonal antibody method described by Chu et al. [23] and adapted by An and Carmichael [24] . Antibody-coated plates , standards, and all reagents were supplied by Abraxis LLC (Warminster, PA). The LOD for the total of all microcystin variants using this method was approximately 0.15 μg /L. Microcystins were quantified using a Stat Fax 303+ spectrophotometer (Envirologix, Portland, ME) at a wavelength of 450 nm in conjunction with a reference wavelength of 630 nm. A final es timate for microcystins content was obtained and calculated as the mean of at least two subs amples (i.e., two replicates per subsample).
High-volume air samplers
To collect large quantities of material for mi crocystin analysis, two po rtable high-volume air samplers (Model TE 5200, Tisch Environmental, In c., Cleves, OH) were placed on a boat. One sampler collected airborne particles in one filter subs trate for total aerosol concentration at a flow rate of 1.5 m 3 /min. For total concentration as well as for particle size dist ribution at a flow rate of 1.2

m 3 /min, the second sampler housed a five-stage , high-volume cascade impactor (Model SA235, Andersen Instruments, Smyrna, Georgia). Cellul ous filters (Filter Paper 41, Whatman International Ltd., Maidstone, UK) were used for stage substrates and backup collection of the samplers. The boat remained stationary, near where water samples we re taken. The sampling time usually started between 8 to 8:30 a.m. and ended between 3:30 to 4:30 p.m.Mar. Drugs 2008, 6 401

Personal Samplers
Personal exposure was measured using a personal sampler connected to a battery-operated pump (SKC, Inc., Eighty Four, PA). If the participant had activities on th e boat or beach, the sampler was placed at the lapel near the breathing zone. If par ticipants were in the same recreational group, a single sampler was placed on their boat. A 25-mm cellulo se filter (Filter Paper 41, Whatman International Ltd., Kent, UK) was used as the collection subs trate. The sampling flow rate was 10.6 L min -1 controlled by the sampling pump.
Analysis of Air Samples
The analysis of air sample s is described by Cheng et al. [25]. Briefly, after collection, the highvolume impactor substrates were stored at -20o C. Filters were removed from cold storage and before extraction, equilibrated to room temperature. The w hole filter of a personal sampler or a piece of 3.0

cm 2 cut from the high-volume sampler substrate was placed in a glass vial, and 1 mL of phosphatebuffered saline was added. The vial was pl aced on a circular rotator (Roto-Torque ® Low Speed #10; Cole-Parmer Instruments, Vernon Hills, IL) for 10 min. After rotation, the filte r was removed from the extraction tube and the extract was used for ELI SA (ELISA kits for microcystin-LR, Production #520011; Abraxis LLC, Warminster, PA) analysis of microcystin. Before analysis—to account for any solution absorbed by the filter—the final volume of the remaining extraction solution was measured. The limit of detection of the microcystin using the ELISA assay was 70 ng/L. For total particle highvolume filter samplers, this corresponded to about 0.0037 ng/m 3

. Waterborne pathogens

We collected 24, 10-L lake water sa mples at the same locations and times as the samples for algal taxonomy and toxin analyses and concentrated the sa mples for molecular analysis. Each sample was concentrated using an ultraf iltration procedure based on Hill et al. [26]. Ultrafiltration concentrates (~ 400 mL each) were shipped overnight in chilled cool ers to the laboratory at the Centers for Disease Control and Prevention (CDC) in Atlanta. We furthe r concentrated viruses us ing polyethylene glycol (PEG) precipitation (addition of 8% PEG 8000, 0.3M NaCl, and 1% Bovine Serum Albumin; incubation overnight at 4o C). Final PEG concentrates were ~ 5 mL in volume. DNA and RNA were simultaneously extracted from a volume of 500 μL from each sample [27]. Final nucleic acid extracts were ~100 μL, and 20-μL volumes of nucleic acid ex tract were used for each PCR and RT-PCR assay. Each sample was assayed for adenoviruses using a broadly reactive real-time PCR assay [28] and a pan-enterovirus real-time RT-PCR assay [29]. PCR i nhibition was evaluated in each of the 24 samples by seeding a reagent-grade water c ontrol and an aliquot of DNA extr act from each sample with the same amount of DNA from a stock of adenovirus 40, an d testing these seeded specimens and controls using the adenovirus real-time PCR assay. Differen ces in CT values between controls and water samples were used as indications of the magnitude of PCR inhibition of the adenovirus assay. This approach to evaluating PCR inhibition for water samples has been previously reported [27].Mar. Drugs 2008, 6 402

Study participants
During August 4–6, 2006, we recruited study particip ants on-site. Specifica lly, we approached approximately 133 people who came to the study lakes for recreational ac tivities. Of these, approximately 29 (22%) refused for va rious reasons, including that they were either too busy (13), not interested (4), afraid of giving a blood sample (4), or not planning to swim in the lake (5). The remaining three people did not report why they were not interested. Interested individuals read and signed a consent form. They were interviewed on th e day of the study in pe rson and later by telephone. After their recreati onal activities, each provide d a blood sample that was analyzed for microcystin concentrations.
Questionnaires
We conducted personal interviews to collect de mographic data (e.g., age, sex, race) and selfreported symptom data including flu-like symptoms (f ever, chills, sort throat, headache, earache), eye irritation (blurred vision, eye irri tation, red eyes, conjunc tivitis), dermatologic symptoms (itchy skin, red skin, hives, skin irritation, rash, infections), respiratory sy mptoms (cough, shortness of breath, congestion, throat irritation) , neurologic symptoms (agitati on, confusion, dizziness, lethargy, weakness, seizure, numbness, tremor), and gastro intestinal symptoms (abdominal pain, nausea, vomiting, diarrhea). We asked whether participants had any of the symp toms during the week before the study as well as before and after recreational activities on the lake s. Using a telephone interview, approximately 10–14 days after exposure we again coll ected self-reported symptom data. We used SAS/STAT software Version 9.1 (SAS In stitute, Cary, North Carolina) for statistical analyses. For each participant in each time peri od individual symptoms were summed and then dichotomized to represent any-versus-no symptoms. We used Fisher’s Exact Test [30] to examine whether people in the exposed group reported more frequently having any symp toms than did those in the control group.
Blood sample analysis
Methods and parameters used in this study for analysis of microcystins in human blood samples were based on two human exposure episodes in Braz il—one in 1996 and the more recent in 2004 [5, 6, 31, 32, 33]. While both of these events occurred becau se of contaminated drinking water used in a kidney dialysis facility, the analys is methods are relevant to the current study where exposure resulted from swallowing water or inhaling spray dur ing water-based recreational activities. Blood samples were collected in 6-ml Vacu tainer® tubes (Becton, Dickenson, and Company, Franklin Lakes, New Jersey) containing sodium heparin and then centrifuged. The plasma samples were shipped frozen and stored at -70 o C. Blood plasma (0.5 ml) was ex tracted in 10 ml of methanol. The extract was centrifuged at 7,500 rpm for 15 minutes. The supernatan t was transferred into a flask and the pellet was re-extracted in 5-ml methanol and re-centrifuged. The supernatants were combined, and 30 ml of hexane was added. The flask was shak en and the hexane layer removed. The methanol extract was dried down and reconstituted in 6 mL of 5% metha nol in 0.1 M acetic acid. The sample was passed through a Waters Oasis HLB® extracti on cartridge (Waters Cor poration, Milford, MA).Mar. Drugs 2008, 6 403

The cartridge was washed with 2 mL of 30% metha nol in 0.1 M acetic acid. Microcystins were eluted with 10 mL of methanol. This fraction was saved a nd dried down. The fraction was reconstituted in 1 mL of 20% MeOH and centrifuged for 10 minutes at 1400 x g. The supernatan t was passed through a Microcon® YM-10 (10,000 MW cutoff) (Millipore, B illeria, MA) centrifugal filter. The filtrate was used for analysis. For the microcystins each sample was analyzed using an Enzyme-Linked Immunosorbent Assay (ELISA). The ELISA method was based upon the or iginal polyclonal antibody method described by Chu et al. [1990] and adapted by An and Carmichael [24] and Carmichael and An [34]. The limit of quantification (LOQ) for microcys tin on the ELISA plate is 0.175 μg/L. The limit of detection (LOD) for microcystin on the ELISA plate is 0.147 μg/L. Samples were run on a Molecular Devics Corp. (Palo Alto, CA), Vmax ki netic microplate reader. For the single blood sample that was positive (above the limit of detection for the ELISA) for microcystins, electrospray ionization mass spect rometry (ESI/MS) and MS/MS analyses were determined using a Finnigan Thermo-Quest LCQ Duo benchtop LC/MS/MS. The LCQ Duo MS detector was operated using the posit ive ESI mode run at a 4.0 kV sp ray voltage. The signals produced were analyzed using Xcalibur software versi on 1.1 (ThermoFinnigan, San Jose, CA). Microcystins were separated on a C18 ODS (4 mm x 2 mm i.d.) guard cartridge (Phenome nex, Torrance, CA) and a Pursuit C18 3μ PN 3001 – 100 x 020 column (Varian, Palo Alto, California) at a flow rate of 0.2 mL/min. The mobile phase was (A) HPLC grade water containing 0.1 M HOAc and (B) acetonitrle containing 0.1 M HOAc. For microcystin-LR, the ma ss spectrometer was operated in the full scan mode for MS/MS/-mlz 270-1050. For microcystin-RR, the mass spectrometer was operated in the full scan mode for MS/MS-mlz 285-1080. For microcystin-Y R, the mass spectrometer was operated in the full scan mode for MS/MS-mlz 285-1100. The limits of detection and quantification for microcystinLR, -RR, and -YR by LC/MS without concentr ation are 0.1, 1, and 10 μg/L, respectively. In summary, we collected usable blood samples from 96 exposed and 6 unexposed participants. Of the 102 blood samples, 101 samples tested below the limit of detection acco rding to the limit of detection described in the Enviro logix Kit (0.147 μg/L). Only one blood sample showed detectable levels of microcystins—about 1.0 μg/L. LC/MS anal ysis of this sample showed the absence of microcystin-LR, -RR, and -YR. Given the LC/MS resu lts, we concluded that this sample represents either a false positive or exposure from microcystin(s) not identified as microcystin-LR, -RR, or -YR.
Acknowledgments
The authors would like to acknowledge Jane Hort on of the National Center for Environmental Health, Dr. Gary Fahnenstiel, and Dennis Dona hue of the National Oceanic and Atmospheric Administration; Dr. Richard R. Rediske of Grand Valley State University, Alexyz Milian , David Jayroe of Mote Marine Laboratory , and Augie and Peg Kotlewski for their invaluable assistance in conducting this study.

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