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

Marine Drugs

ISSN 1660-3397

www.mdpi.org/marinedrugs

Article 

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, Centers for Disease Control and Prevention, 4770 

Buford Highway NE, Chamblee, Georgia 30341, USA

2 Department of Biological Sciences, Wright State University, 3640 Colonel Glen Highway, Dayton, 

Ohio 45435. Current address: 42184 Tweedle Lane, Seaside, Oregon 97138, USA

3 Mote Marine Laboratory, 1600 Ken Thompson Parkway, Sarasota, Florida 34236, USA

4 Greenwater Laboratories, 205 Zaegler 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 Foundation, 1670 Clairmont Road, Decatur, Georgia 30033, USA

* Author to whom correspondence should be addressed; E-mail: lbacker@cdc.gov; Tel: +1-770-488-

3426; Fax: +1-770-488-3450. 

Received: 6 February 2008; in revised form: 5 June 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, jet 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 recreational activities in that lake and seven 

others who volunteered to recreate in a nearby bloom-free lake. We conducted our field 

study within a week of finding a 10-μg/L microcystin concentration. 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 information. Water samples were assayed for 

potential respiratory viruses (adenoviruses and enteroviruses), but none were detected. We 

did find low concentrations of Escherichia coli, indicating fecal contamination. We found 
Mar. Drugs 2008, 6 390

low levels of microcystins (2 μg/L to 5 μg/L) in the water and (<0.1 ng/m3

) 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 microcystins. This is the first study to report 

that water-based recreational activities can expose people to very low concentrations of 

aerosol-borne microcystins; we recently conducted 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 with cyanobacterial (blue-green algal) HAB toxins, 

or with failures to protect water used for medical purposes, such as kidney dialysis [5, 6]. 

In addition to consuming toxins directly in contaminated 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 marine aerosols and blown onshore [7]. 

When these aerosols are inhaled, they induce respiratory symptoms in beach visitors and in healthy 

lifeguards [8, 9] and detectable changes in pulmonary function in people with asthma [10, 11]. 

Stewart et al. [12] reviewed case reports and anecdotal references that describe illnesses associated 

with exposure to cyanobacteria and related toxins during recreational activities in freshwater 

environments. The reported symptoms included hay-fever-like symptoms, gastroenteritis, pyritic skin 

rashes, and allergic reactions. Others, however, reported more severe acute reactions including severe 

headaches, fever, and blistering in the mouth. 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 respiratory irritation. In addition to these case reports and anecdotes, 

epidemiologic studies examining recreational exposures to cyanobacteria have been published [13, 14, 

15, 16, 17]. Most studies did not find an association between recreational exposure 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 who were exposed to waters that did not contain 

cyanobacteria (odds ratio = 3.44; 95% confidence 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, or 
Mar. Drugs 2008, 6 391

Florida (US). In the follow-up phone interview conducted at least 3 days following the water-contact 

activities, people who used personal watercraft on lakes with high levels of cyanobacteria (cell surface 

area >12.0 mm2

/mL) were 2.1 (CI, 1.1 – 4.0) times more likely to report symptoms—particularly 

respiratory symptoms—than were people who used their personal watercraft on lakes with low levels 

of cyanobacteria (cell surface area < 2.4 mm2/mL). 

Using available evidence that supports an association between exposure to waters containing high 

concentrations of cyanobacteria and subsequent adverse health effects, many entities, including the 

World Health Organization (WHO), Australia, and some other European countries, have developed 

guidelines for managing recreational waters with cyanobacterial blooms [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 probability 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 algal scum appears on the water surface. 

These guidelines are based on cell concentrations rather 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 limiting access to recreational waters. Epidemiologic studies to assess 

symptoms and to measure concentrations of cyanobacterial toxins in the environment (water and 

aerosols) could provide data needed 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 watercraft) in a lake during a microcystinproducing algal bloom. In addition, we were interested in determining whether microcystin 

concentrations in aerosol and water samples were associated with microcystin concentrations in blood 

and with self-reported symptoms. 

Results and discussion 

Study site 

We were aware of a number of small recreational lakes in Michigan, New York, Ohio, and other states 

that historically experienced microcystin-producing algal blooms in late summer, and we corresponded 

with colleagues conducting routine monitoring of these lakes. Based on previous experience with 

accidental exposures to water contaminated with microcystins, 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 detectable in blood was about 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 study 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 changes in local weather can, however, rapidly change algal bloom 

characteristics. For this field study, water sample analyses completed after the study found that even 
Mar. 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 microcystin in blood samples 

from our study subjects. Nevertheless, in this field study we verified that inhalation is a possible route 

of exposure to microcystins, and these 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 sites, each sampled in the morning and afternoon, on 

each of the 3 study days, August 4-6, 2006) for analysis of water quality, alga taxa, and microcystin 

concentrations from the lake with the algal bloom. Table 1 contains the ranges of water quality 

parameters tested over the 3-day study period as well as weather data collected during that same 

period. 

In general, cyanobacterial populations are found and thrive in warm, calm waters with high 

concentrations of nutrients and characterized by consistent, relatively 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 prerequisite for growth. Still, if potentially toxigenic species were present, 

warm temperatures (25–30o

 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 the aquatic environment. Under normal environmental conditions in a 

healthy, reproductively active toxin-producing bloom event, 95% to 98% of the toxins would be 

intracellular. Accordingly, we would expect to find 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 accumulations and diluting microcystin 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 four samples sites where 

water samples were collected were open water—thus we would not expect high 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 and found microcystin concentrations of 43 ug/L. 

Later that morning and in the afternoon we did not observe cell accumulations, and in the water 

samples, toxin concentrations remained relatively 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 stations. 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. Drugs 2008, 6 

Table 2. Densities of potentially toxigenic cyanobacteria in water samples from four sampling stations o

with the algal bloom. 

Potentially Toxigenic Cyanobacteria 

(PTOX) (cells/mL) 

Station 1 Station 2 Stat

Genera Species Day 1 Day 2 Day 3 Day 1 Day 2 Day 3 Day 1 Da

Anabaena Macrospore 4,925 5,746 11,492 1,437 10,056 7,183 1,437 456  Spiroides 2,062 3,932 983 983 2,949 2,873 3,932 286  Sp. 5,444 6,351 12,702 3,969 11,432 3,811 6,351 2,72Aphanizomenon Aphanizomenonoides/ 

gracile 

25,923 24,295 18,146 24,195 25,404 39,921 18,146 2,59

 Issatschenkoi 7,777 3.024 3,024 2,268 3,629 2,268 3,024 756 

Cf. flos-aquae 5,444 3,176 9,527 6,351 7,621 11,432 3,176 2,72

Cylindrospermopsis Raciboskii 0 0 1,361 340 0 3,266 0 1,16Microcystis Cf. botrys 20,220 5,897 15,726 5,897 18,872 37,744 7,863 6,74 Wesenbergii 3,213 1,870 3,213 12,853 2,210 6,427 1,190 85  Sp. 6,049 240 6,049 240 80 29,033 80 480 

Total PTOX cells/mL 81,057 54,431 82,223 58,533 82,253 143,958 45,199 18,0
Mar. Drugs 2008, 6 395

Table 2 contains the densities of potentially toxigenic cyanobacteria (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-producing 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 collected water samples any potential relationship between 

the number of microcystin-producing PTOX-C and 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/m3

) on each stage of the high volume, airborne particle impactor. The flow rate, sampling 

time, and total concentration of microcystins from the five stages and the back-up filters, respectively, 

were as follows: day 1, 1.17 m3

/min, 360 min, 0.050 ng/m3; day 2, 1.15 m3/min, 386 min, 23 ng/m3; 

and day 3, 1.22 m3

/min, 484 min, 0.057 ng/m3. 

We deployed 10 personal air samplers on study days 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 microcystins using personal air samplers. 

Table 3. Mass and air concentrations of microcystins collected on each study day using 

personal samplers carried by study participants or placed on their boats. 

Study 

Day 

Mean 

Sampling 

Time (min) 

Mass of microcystins on filter (ng) Air concentration of microcystins 

(ng/m3) 

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 flow rate was 0.0106 m3/min. 

Waterborne pathogens 

Stewart et al. [18] found an increase in respiratory symptoms in people exposed to higher levels of 

cyanobacteria (surface area > 12.0 mm2

/mL) than in people exposed to lower levels of cyanobacteria 

(surface area < 2.4 mm2/mL). It is possible that respiratory symptoms could be caused by infections 

from viruses rather than cyanobacteria. Thus, for our study, we added the analysis of the presence of 

adenoviruses, as well as enteroviruses in the lake water. 
Mar. Drugs 2008, 6 396

 Each 10-L water sample was tested for adenoviruses using real-time PCR and enteroviruses using 

real-time reverse-transcription (RT)-PCR. Adenoviruses and enteroviruses 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-forming units (PFU) per 10-L sample. Thus, these data 

indicate that adenoviruses and enteroviruses were not present at substantial concentrations 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 seeded with an inhibitor standard 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 constituents in the water samples—such as algal matter 

and other organic material (including humic and fulvic acids)—that are generally reflected by the 

relatively high turbidity data 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 quality indicator that has been shown to 

correlate with reports of gastrointestinal illness in people exposed to the water during recreational 

activities [21, 22]. Thus despite the absence of any fecal-associated virus contamination 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 visitors planning recreational 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. Ninety-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-seven (59%) reported that during the 7 

days before the study they had participated in water-related recreational activities on the study lake. In 

addition, these exposed participants reported that in the year before 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 exposed group reported the following activities during the study period: 

swimming (73 [76%]), boating (34 [51%]), tubing (20 [30%]), riding personal 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 time ranged from 15 min to 120 min. Of those who did the other activities, the amount of time 

ranged from 10 min to 300 min Sixty-one (64%) of the exposed group reported 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-Hispanic, and 4 (57%) were female. Of the 7 unexposed participants, 

6 (86%) reported they participated in water-related recreational activities on the study lake (exposed 
Mar. 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 reported only swimming during 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, and 1 (14%) reported swallowing 

water. 

Table 4 contains a summary of symptoms reported by both exposed and unexposed participants 

during the interviews. As a group, study participants reported more symptoms in the 7 days before the 

study than during study or during the 7 to 10 days after the study. In addition, as a group, study 

participants tended to report more symptoms (e.g., cough, sore throat for the exposed group, skin 

complaints for the unexposed group) immediately before doing study activities than immediately after 

such activities. A single participant in the control group reported five skin-related symptoms before 

going into the water but not after coming out of the water. No differences appeared in the frequency of 

reported symptoms between the exposed and control groups during the 7 days before the study, 

immediately before or immediately after doing study activities, or during the follow-up period. 

We hypothesized that exposure to aerosolized microcystins would result in acute dermal and 

respiratory symptoms; thus, we expected people to report more symptoms immediately after study 

activities than during the week before the study. However, more people reported symptoms before 

doing study activities than after. In addition, participants reported more symptoms for the period of 7 

days before the study than either immediately before or after doing study activities. 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-related recreational activities 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 immediately 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 days after the exposure, i.e., during the week after 

weekend activities on the blooming lake. This possibility 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 symptom surveys before their first visit to the 

lake and then periodically throughout the summer. 

Blood samples 

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 according to the limit of detection 

described in the Envirologix Kit (0.147 μg/L). Only one blood sample showed detectable 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 results, 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 supplements). 
Mar. Drugs 2008, 6 398

Table 4. Self-reported symptom data from study participants engaged in recreational 

activities in a lake with an algal 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 

study 

activities 

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 study activities, immediately 

before and after doing study activities, and during the telephone follow-up interview. Symptoms listed 

are those reported by more than two 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 Institutional Review Board of the Centers for Disease Control and 

Prevention. 

Environmental sampling sites 

We used four sampling sites located along the major axis of a lake that had historical water quality 

data previously collected by Grand Valley State University. Each time sampling was performed 

latitude and longitude coordinates 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 present. Algal samples were collected at a depth of 0.15 meters. Water 

samples used for algal identification were immediately preserved with 10% Lugols solution and stored 

under dark, cool conditions. 

Water samples were collected for microcystin analyses at a depth of 0.15 meters by directly 

immersing sample containers in ambient water. Before 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 conditions (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 study was completed. 

For algal identification and microcystin analyses, general water quality parameters (e.g., water 

temperature, dissolved oxygen, pH, specific conductivity, salinity, and turbidity) 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 data sonde (Hacj Company, Loveland, CO) supplied 

by Dr. R. Rediske of Grand Valley State University. Water quality parameters were measured at a 

depth of 0.15 meters. At the time of water collection ambient environmental conditions (cloud cover, 

wind speed, wind direction, and air temperature) were recorded at each site. Grand Valley State 

University performed water chemistry on the morning of August 4th, 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 bottle 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 base 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 settling tower. A cover glass was placed on top of the tower, and the 
Mar. Drugs 2008, 6 400

sample was allowed to settle in the dark in a vibration-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 covered with a second cover glass. 

Enumeration was performed on a Nikon Eclipse TE200 (Southern Microscopes, Inc., Mebane, NC) 

inverted microscope equipped with phase-contrast optics. 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. Before 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 both. 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 4o

C for 

toxin analyses. Microcystin analyses were performed within 24-48 hours of receipt of samples. 

Enzyme linked immunosorbent assay (ELISA) was utilized for the determination 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 estimate for microcystins content was obtained and 

calculated as the mean of at least two subsamples (i.e., two replicates per subsample). 

High-volume air samplers 

To collect large quantities of material for microcystin analysis, two portable high-volume air 

samplers (Model TE 5200, Tisch Environmental, Inc., Cleves, OH) were placed on a boat. One 

sampler collected airborne particles in one filter substrate for total aerosol concentration at a flow rate 

of 1.5 m3

/min. For total concentration as well as for particle size distribution at a flow rate of 1.2 

m3/min, the second sampler housed a five-stage, high-volume cascade impactor (Model SA235, 

Andersen Instruments, Smyrna, Georgia). Cellulous 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 were 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 the boat or beach, the sampler was 

placed at the lapel near the breathing zone. If participants were in the same recreational group, a single 

sampler was placed on their boat. A 25-mm cellulose filter (Filter Paper 41, Whatman International 

Ltd., Kent, UK) was used as the collection substrate. The sampling flow rate was 10.6 L min-1

controlled by the sampling pump. 

Analysis of Air Samples 

The analysis of air samples 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 whole 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 placed on a circular rotator (Roto-Torque® Low Speed #10; 

Cole-Parmer Instruments, Vernon Hills, IL) for 10 min. After rotation, the filter was removed from the 

extraction tube and the extract was used for ELISA (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/m3

. 

Waterborne pathogens 

We collected 24, 10-L lake water samples at the same locations and times as the samples for algal 

taxonomy and toxin analyses and concentrated the samples for molecular analysis. Each sample was 

concentrated using an ultrafiltration procedure based on Hill et al. [26]. Ultrafiltration concentrates (~ 

400 mL each) were shipped overnight in chilled coolers to the laboratory at the Centers for Disease 

Control and Prevention (CDC) in Atlanta. We further concentrated viruses using polyethylene glycol 

(PEG) precipitation (addition of 8% PEG 8000, 0.3M NaCl, and 1% Bovine Serum Albumin; 

incubation overnight at 4oC). 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 extract 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 inhibition was evaluated in each of the 24 samples 

by seeding a reagent-grade water control and an aliquot of DNA extract from each sample with the 

same amount of DNA from a stock of adenovirus 40, and testing these seeded specimens and controls 

using the adenovirus real-time PCR assay. Differences 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 participants on-site. Specifically, we approached 

approximately 133 people who came to the study lakes for recreational activities. Of these, 

approximately 29 (22%) refused for various 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 the day of the study in person and later by telephone. 

After their recreational activities, each provided a blood sample that was analyzed for microcystin 

concentrations. 

Questionnaires

 We conducted personal interviews to collect demographic data (e.g., age, sex, race) and selfreported symptom data including flu-like symptoms (fever, chills, sort throat, headache, earache), eye 

irritation (blurred vision, eye irritation, red eyes, conjunctivitis), dermatologic symptoms (itchy skin, 

red skin, hives, skin irritation, rash, infections), respiratory symptoms (cough, shortness of breath, 

congestion, throat irritation), neurologic symptoms (agitation, confusion, dizziness, lethargy, 

weakness, seizure, numbness, tremor), and gastrointestinal symptoms (abdominal pain, nausea, 

vomiting, diarrhea). 

We asked whether participants had any of the symptoms during the week before the study as well as 

before and after recreational activities on the lakes. Using a telephone interview, approximately 10–14 

days after exposure we again collected self-reported symptom data. 

We used SAS/STAT software Version 9.1 (SAS Institute, Cary, North Carolina) for statistical 

analyses. For each participant in each time period 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 symptoms 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 Brazil—one in 1996 and the more recent in 2004 [5, 6, 

31, 32, 33]. While both of these events occurred because of contaminated drinking water used in a 

kidney dialysis facility, the analysis methods are relevant to the current study where exposure resulted 

from swallowing water or inhaling spray during water-based recreational activities. 

Blood samples were collected in 6-ml Vacutainer® tubes (Becton, Dickenson, and Company, 

Franklin Lakes, New Jersey) containing sodium heparin and then centrifuged. The plasma samples 

were shipped frozen and stored at -70o

 C. Blood plasma (0.5 ml) was extracted in 10 ml of methanol. 

The extract was centrifuged at 7,500 rpm for 15 minutes. The supernatant 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 shaken and the hexane layer removed. The methanol 

extract was dried down and reconstituted in 6 mL of 5% methanol in 0.1 M acetic acid. The sample 

was passed through a Waters Oasis HLB® extraction cartridge (Waters Corporation, Milford, MA). 
Mar. Drugs 2008, 6 403

The cartridge was washed with 2 mL of 30% methanol in 0.1 M acetic acid. Microcystins were eluted 

with 10 mL of methanol. This fraction was saved and dried down. The fraction was reconstituted in 1 

mL of 20% MeOH and centrifuged for 10 minutes at 1400 x g. The supernatant was passed through a 

Microcon® YM-10 (10,000 MW cutoff) (Millipore, Billeria, 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 original 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 microcystin 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 kinetic microplate reader. 

For the single blood sample that was positive (above the limit of detection for the ELISA) for 

microcystins, electrospray ionization mass spectrometry (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 positive ESI mode run at a 4.0 kV spray voltage. The signals produced 

were analyzed using Xcalibur software version 1.1 (ThermoFinnigan, San Jose, CA). Microcystins 

were separated on a C18 ODS (4 mm x 2 mm i.d.) guard cartridge (Phenomenex, 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 mass 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-YR, 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 concentration 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 according to the limit of 

detection described in the Envirologix Kit (0.147 μg/L). Only one blood sample showed detectable 

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 results, 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 Horton of the National Center for Environmental 

Health, Dr. Gary Fahnenstiel, and Dennis Donahue 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. 

References 

1. Baden, D.G.; Trainer, V.L. Mode of action of toxins of seafood poisoning. In Algal Toxins in 

Seafood and Drinking Water; Falconer, I.R., Ed.; Academic Press: London, UK, 1993; pp. 49-74. 
Mar. Drugs 2008, 6 404

2. Carmichael, W.W.; Falconer, I.R. Diseases related to freshwater blue-green algal toxins, and 

control measures. In Algal Toxins in Seafood and Drinking Water; Falconer, I.R., Ed.; Academic 

Press: London, UK, 1993; pp. 187-209. 

3. Steidinger, K.A. Some Taxonomic and biologic aspects of toxic dinoflagellates. In Algal Toxins in 

Seafood and Drinking Water; Falconer, I.R., Ed.; Academic Press: London, UK, 1993; pp. 1-28. 

4. Backer, L.C. Cyanobacterial harmful algal blooms (CyanoHABs): Developing a public health 

response. Lake Reserv. Manag. 2002, 18, 20-31. 

5. Billings, W.H. Water-associated human illness in northeast Pennsylvania and its susptected 

association with blue-green algae blooms. In The Water Environment: Algal Toxins and Health; 

Carmichael, W.W., Ed.; Plenum Press: New York, NY, USA, 1981; pp. 243-255. 

6. Falconer, I.R.; Beresford, A.M.; Runnegar, M.T.C. Evidence of liver damage by toxin from a 

bloom of the blue-green alag, Microcystis aeruginosa. Med. J. Aust. 1983, 1, 511-514. 

7. Hayman, J. Beyond the Barcoo—probable human tropical cyanobacteria poisoning in outback 

Australia. Med. J. Aust. 1992, 157, 794-796. 

8. Pilotto, L.S.; Kliewer, E.V.; Davies, R.D. Cyanobacterial (blue-green algae) contamination in 

drinking water and perinatal outcomes. Aust. N. Z. J. Pub. Health 1999, 23, 154-158. 

9. Jochimsen, E.M.; Carmichael, W.W.; An, J.S.; Cardo, D.M.; Cookson, S.T.; Holmes, C.E.M.; 

Antunes, M.B.de C.; Filho, D.A. de Melo; Lyra, T.M.; Burreto, V.S.T.; Azevedo, S.M.F.O.; 

Jarvis, W.R. Liver failure and death after exposure to microcystins at a hemodialysis center in 

Brazil. New Eng. J. Med. 1998, 338, 873-878. 

10. Carmichael, W.W.; Azevedo, M.F.O.; An, J.S.; Molica, R.J.R., Jochmisen, E.M., Lau, S.; 

Rinehart, K.L.; Shaw, G.R.; Eagelsham, G.K. Human fatalities from cyanobacteria: chemical and 

biological evidence for cyanotoxins. Environ. Health Perspect. 2001, 109, 663-668. 

11. Pierce, R.H.; Henry, M.S.; Proffitt, L.S.; Hasbrouck, P.A. Red tide toxin (brevetoxin) enrichment 

in marine aerosol. In Toxic Marine Phytoplankton; Graneli, E., Sundstrom, B., Elder, L., 

Anderson, D.M., Eds.; Elsevier: Amsterdam, Netherlands, 1990; pp. 397-402. 

12. Backer, L.C.; Fleming, L.E.; Rowan, A.; Cheng, Y.-S.; Benson, J.; Pierce, R.H.; Zaias, J.; Bean, 

J.; Bossart, G.D.; Johnson, D.; Quimbo, R.; Baden, D.G. Recreational exposure to aerosolized 

brevetoxins during Florida red tide events. Harmful Algae 2003, 2, 19-28. 

13. Backer, L.C.; Kirkpatrick, B.; Fleming, L.E.; Cheng, Y.S.; Pierce R.; Bean, J.A.; Clark R.; 

Johnson, D.; Wanner, A.; Tamer, R.; Zhou, Y.; Baden, D.G. Occupational exposure to aerosolized 

brevetoxins during Florida red tides: effects on a health worker population. Environ. Health 

Perspect. 2005, 113, 644-649. 

14. Fleming, L.E.; Kirkpatrick, B.; Backer, L.C.; Bean, J.A.; Wanner, A.; Dalpra, D.; Tamer, R.; 

Zaias, J.; Cheng, Y.S.; Pierce, R.; Naar, J.; Abraham, W.; Clark, R.; Zhou, Y.; Henry M.S.; 

Johnson, D.; Van De Bogart, G.; Bossart, G.; Harrington, M.; Baden, D.G. Initial evaluation of the 

effects of aerosolized Florida red tide toxins (brevetoxins) in persons with asthma. Environ. 

Health Perspect. 2005, 113, 650-657. 

15. Fleming, L.E.; Kirkpatrick, B.; Backer, L.C.; Bean, J.A.; Wanner, A.; Reich, A.; Dalpra, D.; 

Zaias, J.; Cheng, Y.S.; Pierce, R.; Naar, J.; Abraham, W.M.; Baden, D.G. Aerosolized Red-Tide 

Toxins (Brevetoxins) and Asthma. Chest 2007, 131, 187-194. 
Mar. Drugs 2008, 6 405

16. Stewart, I.; Webb, P.M.; Schluter, P.J.; Shaw, G.R. Recreational and occupational field exposure 

to freshwater cyanobacteria—a review of anecdotal and case reports, epidemiologic studies, and 

the challenges for epidemiologic assessment. Environ. Health 2006a, 5, 6. 

17. Phillip R. Health risks associated with recreational exposure to blue-green algae (cyanobacteria) 

when dinghy sailing. Health Hyg. 1992, 13, 110-114. 

18. Phillip, R.; Bates, A.J. Health-risks assessment of dinghy sailing in Avon and exposure to 

cyanobacteria (blue-green algae). J. Inst. Water Environ. Manage. 1992, 6, 613-620. 

19. Phillip, R.; Brown, M.; Bell, R.; Francis, F. Health risks associated with recreational exposure to 

blue-green algae (cyanobacteria) when windsurfing and fishing. Health Hyg. 1992, 13, 115-119. 

20. El Saadi, O.E.; Esterman, A.J.; Cameron, S.; Roder, D.M. Murray River water, raised 

cyanobacterial cell counts, and gastrointestinal and dermatological symptoms. Med. J. Aust. 1995, 

162, 122-125. 

21. Pilotto, L.S.; Douglas, R.M.; Burch, M.D.; Cameron, S.; Beers, M.; ROuch, G.J.; Robinson, P.; 

Kirk, M.; Cowie, C.T.; Hardiman, S.; Moore, C.; Attewell, R.G. Health effects of exposure to 

cyanobacteria (blue-green algae) during recreational water-related activities. Aust. N. Z. J. Public 

Health 1997, 21, 562-566. 

22. Stewart, I; Webb, P.M.; Schluter, P.J.; Fleming, L.E.; Burns, J.W. Jr.; Gantar, M., Backer, L.C.; 

Shaw, G.R. Epidemiology of recreational exposure to freshwater cyanobacteria—an international 

prospective cohort study. BMC Pub. Health 2006b, 6, 93. 

23. WHO. Guidelines for safe recreational water environments—Volume 1: coastal and fresh waters; 

World Health Organization: Geneva, Switzerland, 2003; available online: http://www.who.int/ 

water_sanitation_health/bathing/srwe1/en/. 

24. Chorus, I. (Editor). Current approaches to cyanotoxin risk assessment, risk management and 

regulation in different countries; German Federal Environmental Agency (Umweltbundesamt): 

Berlin, Germany, 2005; available online: http://www.umweltdaten.de/publikationen/fpdf-l/ 

2910.pdf. 

25. United States Environmental Protection Agency (EPA). Ambient Water Quality Criteria for 

Bacteria—1986; Office of Water, EPA440/5-84-002, 1986. 

26. Wade, T.J.; Calderon, R.C.; Sams, E.; Beach, M.; Brenner, K.P.; Williams, A.H.; Dufour, A.P. 

Rapidly measured indicators of recreational water quality are predictive of swimming-associated 

gastrointestinal illness. Environ. Health Perspect. 2006, 114, 24-28. 

27. Chu, F.S.; Huang, X.; Wei, R.O. Enzyme-linked immunosorbent assay for microcystins in bluegreen algal blooms. J. Assoc. Off. Analyt. Chem. 1990, 73, 451-456. 

28. An, J-S.; Carmichael, W.W. Use of a colorimetric protein phosphatase inhibition assay and 

enzyme linked immunosorbent assay for the study of microcystins and nodularins. Toxicon 1994, 

32, 1495-1507. 

29. Cheng, Y.S.; Zhou, Y.; Irvin, C.M.; Kirkpatrick, B.; Backer, L.C. Characterization of Aerosols 

Containing Microcystin. Mar. Drugs 2007, 5, 136-150. 

30. Hill, V.R.; Polaczyk, A.L.; Hahn, D.; Jothikumar, N.; Cromeans, T.L.; Roberts, J.M.; Amburgey, 

J.E. Development of a rapid method for simultaneously recovering microbes in drinking water 

using ultrafiltration with sodium polyphosphate and surfactants. Appl. Environ. Microbiol. 2005, 

71(11), 6878-6884. 
Mar. Drugs 2008, 6 406

31. Hill, V.R.; Kahler, A.M.; Jothikumar, N.; Johnson, T.B.; Hahn, D.; Cromeans, T.L. Multistate 

evaluation of an ultrafiltration-based procedure for simultaneous recovery of enteric microbes in 

100-L tap water samples. Appl. Environ. Microbiol. 2007, 73(13), 4218-4225. 

32. Jothikumar, N.; Cromeans T.L.; Hill V.R.; Lu X.; Sobsey M.D.; Erdman D.D. Quantitative realtime PCR assays for detection of human adenoviruses and identification of serotypes 40 and 41. 

Appl. Environ. Microbiol. 2005, 71(6), 3131-3136. 

33. American Water Works Association Research Foundation (AwwaRF). Development of Molecular 

Methods to Detect Infectious Viruses in Water [#2591], 2004; Report No. 90995F. 

34. Sokal, R.R.; Rohlf, F.J. Biometry: The Principles and Practice of Statistics in Biological 

Research. W.H. Freeman and Company: San Francisco, CA, USA, 1981; 735 p. 

35. Azevdo, S.M.F.O; Camichael, W.W.; Jochimsen, E.M.; Rinehart, K.L.; Lau, S.; Shaw, G.R.; 

Eaglesham, G.K. Human intoxication by microcytins during renal dialysis treatment in CaruaruBrazil. Toxicology 2002, 181, 441-446. 

36. Hillborn, E.D.; Carmichael, W.W.; Yuan, M.; Azevedo, S.M.F.O. A simple colorimetric method 

to detect biological evidence of human exposure to microcystins. Toxicon 2005, 46, 218-221. 

37. Soares, R.M.; Yuan, M.; Servaites, J.C.; Delgado, A.; Magalhaes, V.F.; Hilborn, E.D.; 

Carmichael, W.W.; Azevedo, S.M.F.O. Sub-lethal exposure from microcystins to renal 

insufficiency patients in Rio de Janeiro-Brazil. Environ. Toxicol. 2006, 21, 95-103. 

38. Carmichael, W.W.; An, J-S. Using an enzyme linked immunosorbant assay (ELISA) and a protein 

phosphatase inhibition assay (PPIA) for the detection of microcystins and nodularins. Nat. Toxins

1999, 7, 377-385. 

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