Comparative Mammalian Cell Cytotoxicity of Water Concentrates from

ARTICLE pubs.acs.org/est

Comparative Mammalian Cell Cytotoxicity of Water Concentrates from Disinfected Recreational Pools Michael J. Plewa,*,† Elizabeth D. Wagner,† and William A. Mitch‡ †

Department of Crop Sciences and the Center of Advanced Materials for the Purification of Water with Systems, University of Illinois at Urbana
Champaign, Urbana, Illinois 61801, United States ‡ Department of Chemical and Environmental Engineering, Yale University, New Haven, Connecticut 06520, United States ABSTRACT: Disinfection of recreational pools is essential to prevent outbreaks of infectious disease. Despite the health benefits of swimming, recent research demonstrated an association between the application of disinfectants to recreational pools and adverse health outcomes. Pool waters are extreme cases of disinfection that differ in important respects from disinfected drinking waters. Pool waters are continuously exposed to disinfectants over average residence times extending to months. Disinfection byproduct (DBP) precursors in pools include natural humic substances deriving from the tap water source plus inputs from bathers through urine, sweat, hair, skin, and consumer products including sunscreens and cosmetics. This study presents a systematic, chronic in vitro mammalian cell cytotoxicity analysis of different recreational waters with varied environmental conditions that were derived from a common tap water source. Recreational waters were significantly more toxic than their tap water source. Because trihalomethane concentrations are similar between tap waters and pool waters, using trihalomethanes to monitor exposure in epidemiological studies may not be the best metric. Of primary importance for cytotoxicity were illumination conditions. Pools subjected to a combination of ultraviolet light and free chlorine disinfection indoors, or outdoor sunlight exposure exhibited lower cytotoxicity than their indoor counterparts disinfected with free chlorine. Temperature and total organic carbon content, as an indirect measure of DBP precursors, were less important. Previous research on the same samples demonstrated the genotoxicity of an indoor pool disinfected with bromochlorodimethylhydantoin; the cytotoxicity of this sample was confirmed in the present study. While the association of reduced toxicity with illumination indicates that the agents responsible are photolabile, their identity is unclear. As a broad measure of adverse biological responses, cytotoxicity may be a useful metric to gauge the health impacts of alterations in pool operating conditions.

’ INTRODUCTION Swimming is considered a healthy activity and is the second most popular form of exercise in the United States.1 Notwithstanding public pools, by 2007 the number of private swimming pools in the United States increased to 8.4 million.2 The disinfection of recreational pools has been practiced for many years,3 and is essential to prevent outbreaks of disease.4 Yet from a survey of public pools, a majority had management code violations that could lead to infectious disease.5 A recent review articulated the risks associated with recreational pools and enhanced exposures to microbial pathogens and toxic disinfection byproducts (DBPs).6 General health risks associated with exposure to swimming pools are exceedingly difficult to determine because of differences in source water chemistry, disinfection methods, organic loads, and pool environment. Recent studies have raised concerns on adverse health impacts associated with application of disinfectants to recreational pools,7
12 particularly respiratory and skin ailments in children.7,10,11,13,14 These negative outcomes are associated with water contaminants, disinfectants, and DBPs formed from reactions of the disinfectant, bromide/iodide in the source water, or organic r 2011 American Chemical Society

matter from the source water or contributed by bathers.15
18 The biology and chemistry of disinfected recreational waters is exceedingly complex because these waters represent extreme cases of disinfection, featuring disinfectant contact times on the order of months and elevated precursor concentrations.18 Bathers contribute DBP precursors from urine, sweat, hair, skin, and consumer products including cosmetics and sunscreens;12 humic substance precursors are also present in the source water. Nitrogen-rich precursors may be converted into nitrogenous disinfection byproducts (N-DBPs), including organic chloramines, halonitroalkanes, halonitriles, nitrosamines, and nitramines. Elevated genotoxicity and cytotoxicity are associated with many classes of N-DBPs.17,19 The generation of nitrosamines in disinfected water is especially worrisome because of their carcinogenic potency.15,20
27 DBP exposure through Received: December 21, 2010 Accepted: March 23, 2011 Revised: March 16, 2011 Published: April 05, 2011 4159

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Table 1. Description of Recreational Pools, Water Samples, Environmental Conditions, and Concentration Range of Organic Extracts Analyzed for CHO Cell Chronic Cytotoxicity water sample and pool type

sample no.a

TOC (mg/L)b

tap water for all poolsd swimming pool 1, cold

PS3 PS1

1.2 18.1

swimming pool 1, cold

PS7

5.2

swimming pool 2

PS4

124.8

swimming pool 3

PS5

swimming pool 4 hot tub

disinfectant free chlorine UV and free chlorine

location and

total chlorine residual

temperature

(mg/L as Cl2)

concn. factor range c

indoor, 14 °C indoor, 25 °C

1.4 3.7

50
500 10
100 25
100

UV and free chlorine

indoor, 25 °C

1.6

bromochloro-dimethylhydantoin

indoor, 26 °C

2.4

1
50

12.6

free chlorine

indoor, 29 °C

3.4

2.5
50

PS6

33.1

free chlorine

indoor, 36 °C

3.7

2.5
100

PS2

12.1

free chlorine

indoor, 40 °C

2.8

1
50

swimming pool 6

PS8

23.7

free chlorine

indoor, 25 °C

1.7

2.5
40

swimming pool 6

PS9

33.1

free chlorine

outdoor, 20 °C

1.4

25
400

a

Pool sample numbers used in experimental blind. b Total organic carbon. c The concentration range of each pool water extract evaluated. d The city tap water was the source water for all the recreational pools in the study.

drinking water, swimming, and inhalation is associated with the induction of cancer in humans.28
32 Although the first study on the mutagenicity of pool water was published over 30 years ago,33 only recently have studies linked enhanced genotoxic impact with individual swimmers.34 Other studies have determined the occurrence of specific DBPs in recreational waters and correlated these measurements with metrics for toxicity or adverse health outcomes. These efforts were hampered by the wide array of potentially toxic constituents and the vastly different environmental conditions in these waters. Rather than attempting to link specific byproducts to toxic responses, we sought to compare the overall toxic response of recreational waters that were treated with different disinfectants under different conditions. We previously evaluated the genomic DNA damage in mammalian cells for recreational waters employing the same source water, but subjected to different conditions (e.g., disinfectant, temperature, solar exposure).35 The objective of this research was to analyze in vitro chronic cytotoxicity in mammalian cells with the same samples, and to compare these results with the previous genotoxicity results.

’ EXPERIMENTAL SECTION Sample Preparation and Extraction of Organics. Each pool water sample (8
10 L) was collected in a fluorinated highdensity polyethylene container and the temperature was recorded. Total residual chlorine was analyzed by the DPD colorimetric method.36 Total organic carbon (TOC) was analyzed using a Shimadzu TOC analyzer. Water sample characteristics are provided in Table 1. Eight to 10 L of each water sample was extracted into Fisher HPLC grade methyl tert-butyl ether (MtBE) at Yale University within 1 d of sample collection. MtBE was selected as the extraction solvent for two reasons. First, among common laboratory solvents immiscible with water, the hydrophobicity of MtBE (log Kow = 1.4) is close to that of octanol (log Kow = 2.8), commonly considered as a surrogate for the cellular lipid bilayer. In comparison, the log Kow values for methylene chloride and ethyl acetate are 1.2 and 0.9, respectively. Second, MtBE has been employed to extract a range of traditional and emerging disinfection byproducts. For example, MtBE is used as the extraction solvent for chlorinated and brominated trihalomethanes, haloacetonitriles, and chloropicrin by U.S. EPA Method 551.1, and has been used to extract iodinated trihalomethanes and haloacetic acids.37

The chlorine residual was not quenched prior to sample extraction to enable evaluation of the combined toxicity exerted by the disinfectant and byproducts to which cells would be exposed. To evaluate the potential for extraction of a free chlorine disinfectant, a deionized water sample buffered at pH 7 with 20 mM phosphate buffer was spiked with 200 μM free chlorine (14.2 mg/L as Cl2) and extracted as described below. The sample did not exhibit any significant cytotoxicity, indicating that the cytotoxicity observed in most of the samples derived from the byproducts rather than the disinfectants. Aliquots (1 L) were extracted 3 by separatory funnel into 100 mL of MtBE per extraction. MtBE extracts were combined, dried with ACS grade magnesium sulfate (JT Baker), and concentrated by rotary evaporation to 5 mL, transferred to a glass vial, blown down to dryness under N2, and suspended in 1 mL of Acros HPLC grade ethyl acetate. These concentrated extracts were shipped to the University of Illinois, and stored in Supelco micro reaction vessels with PTFE-lined caps at
22 °C under dark conditions. The samples were further concentrated under dry N2 such that the organics in 10 L of original water were concentrated into 100 μL of ethyl acetate. For each experiment, a known volume of the concentrated sample in ethyl acetate was mixed with a known volume of dimethylsulfoxide (DMSO). Ethyl acetate was removed by passing dry N2 at 42 °C over the sample for 1 min. The sample in DMSO was diluted in F12 cell culture medium. Concentrations were calculated as a concentration factor of the organic materials derived from the original water samples. Biological and Chemical Reagents, Chinese Hamster Ovary Cells. General reagents were purchased from Fisher Scientific Co. (Itasca, IL) and Sigma Chemical Co. (St. Louis, MO). Media and fetal bovine serum (FBS) were purchased from Hyclone Laboratories (Logan, UT) or from Fisher Scientific Co. (Itasca, IL). Chinese hamster ovary (CHO) cells are widely used in toxicology. The transgenic CHO cell line AS5238 was derived from CHO K1-BH4; clone 11
4
8 was isolated from AS52 and expresses normal morphology, cell contact inhibition, and a stable chromosome complement with a consistent cell doubling time.39 Cells were grown in Hams F12 medium plus 5% FBS at 37 °C in a humidified atmosphere of 5% CO2. CHO Cell Chronic Cytotoxicity Assay. The CHO cell microplate chronic cytotoxicity assay measures the reduction in cell density as a function of the concentration of the test agent over a 72-h period.17 This calibrated assay demonstrated a direct relationship between the absorbency of the crystal violet dye associated with 4160

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Figure 1. (A) Cytotoxicity concentration
response curve for pool sample 5 illustrating the regression of the data. The response at each concentration was generated from 8
16 independent clones of CHO cells. The determination of the LC50 value is indicated by the arrowed line. (B) A comparison of the cytotoxicity concentration
response curves from all of the water samples analyzed in this study.

the cell and the number of viable cells.40 A 96-well flat-bottomed microplate was used to evaluate a series of concentrations of the pool water organic extracts. The concurrent negative control consisted of eight wells with 3  103 CHO cells plus F12 þ FBS medium. Eight microplate wells served as the blank control consisting of 200 μL of F12 þ FBS medium only. The remaining wells contained 3  103 CHO cells, F12 þ FBS, and a known concentration of an organic extract in a total of 200 μL. The wells were covered with sterile AlumnaSeal and the cells were incubated for 72 h at 37 °C in a humidified atmosphere of 5% CO2. After 72 h, the medium was aspirated, the cells were fixed in methanol for 10 min and stained for 10 min with a 1% crystal violet solution in 50% methanol. The microplate was washed, 50 μL of DMSO/methanol (3:1 v/v) was added to each well, and the plate was incubated at room temperature for 10 min. The microplate was analyzed at 595 nm with a BioRad

microplate reader; the absorbency of each well was recorded. The averaged absorbency value of the blank wells was subtracted from the absorbency data from each well in the microplate. The mean blankcorrected absorbency value of the negative control was set at 100%. The absorbency for each treatment well was converted into a percentage of the negative control. This procedure normalized the data, maintained the variance and allowed the combination of data from multiple microplates. For each organic extract concentration, 8 replicate wells were analyzed per experiment, and the experiments were repeated 2
4. These data were used to generate a concentration
response curve. Regression analysis was applied to each sample concentration
response curve, which was used to calculate a LC50 value (the organic extract concentration that induced a cell density that was 50% of the negative control). The data were transferred from Excel spreadsheets and analyzed using SigmaPlot 4161

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Table 2. Induction of Chronic Cytotoxicity in CHO Cells by the Recreational Pool and Source Tap Water Samples pool water sample and sample number

lowest cytotoxic concn. factora

r2b

LC50 (concn. factor)c

source tap water: PS3

125

0.99

345.6

swimming pool: PS1

10

0.97

60.6

F10, 85 = 60.5; P e 0.001

swimming pool: PS7

50

0.99

90.5

F9, 86 = 38.3; P e 0.001

swimming pool: PS4

12.5

0.96

21.9

F10, 85 = 66.6; P e 0.001

swimming pool: PS5

10

0.99

20.3

F13, 175 = 113.6; P e 0.001

swimming pool: PS6

2.5

0.98

39.8

F16, 135 = 124.7; P e 0.001

hot tub: PS2

15

0.97

21.7

F10, 85 = 62.7; P e 0.001

swimming pool: PS8 swimming pool: PS9

2.5 25

0.94 0.95

24.2 181.4

F13, 162 = 60.1; P e 0.001 F12, 98 = 150.7; P e 0.001

ANOVA test statistic F 8, 47 = 46.3; P e 0.001

a

The lowest cytotoxic concentration was the lowest concentration factor of the organic extracts in the concentration
response curve that induced a significant amount of cytotoxicity as compared to the negative control. b r2 is the coefficient of determination for the regression analysis upon which the LC50 value (%C1/2 value) was calculated. c The LC50 is the sample concentration factor that induced a cell density that was 50% of the negative control.

8.02, SigmaStat 3.1, and Table Curve 4.03 (Systat Software Inc., San Jose, CA). A one-way analysis of variance (ANOVA) test was conducted. If a significant F value of P e 0.05 was obtained, a Holm
Sidak multiple comparison versus the control group analysis was conducted. The power of the test statistic was maintained as g0.8 at R = 0.05. Safety. Manipulations of toxic chemicals were conducted in certified stage-2 containment biological/chemical safety hoods. This project met the requirements of the Division of Research Safety at the University of Illinois.

’ RESULTS AND DISCUSSION Water Samples. The water samples were the same as those collected in our recent study of the induction of genomic DNA damage by recreational waters.35 These waters were from a single tap water source, and were treated with different disinfectants under different conditions (e.g., temperature and solar exposure) (Table 1). This set of samples included pools disinfected with free chlorine, a combination of ultraviolet light and free chlorine, or bromochlorodimethylhydantoin (BCDMH). The indoor pool featuring a combination of ultraviolet light and free chlorine was sampled twice (PS1 and PS7). Other samples included a cold pool (PS5), a warm pool (PS6), and a hot tub (PS2) within the same indoor facility, as well as indoor (PS8) and outdoor (PS9) samples collected during different seasons from the same pool with a retractable dome. A unique feature of these water samples was that the makeup water for all of the pools was supplied from the same tap water source (PS3), which was also analyzed. For all water samples we monitored the TOC, total chlorine residual, and temperature (Table 1). Cytotoxicity of Water Samples. Figure 1A illustrates a concentration
response curve for the CHO cell cytotoxicity for pool water sample 5 (PS5), an indoor swimming pool disinfected with free chlorine (Table 1). The concentration range is expressed as a concentration fold factor compared to the original water sample. The data for each concentration were the average response from 8
24 independent clones and their standard error. CHO cell cytotoxicity concentration
response curves for all of the water samples are presented in Figure 1B. A regression analysis was conducted for each concentration response curve; the r2 values are presented in Table 2. From these regressions the LC50 value for each sample was calculated. The LC50 (%C1/2) is the sample concentration that induced a cell density that was 50% of the negative control. The LC50 for PS5 is illustrated in Figure 1A. The ANOVA analyses and the lowest

concentration that expressed a significant difference from the negative control are presented in Table 2. To directly compare the cytotoxicity for each water sample we calculated a cytotoxicity index value. The cytotoxicity index value was determined as the reciprocal of the LC50 value  1000; a larger value represents a greater cytotoxic potency. This allows for the direct comparison of the cytotoxicity with the genotoxicity of each water sample that was previously published.35 The data expressed a wide range of cytotoxic responses among the water samples. All of the recreational pool water samples were significantly more cytotoxic than the tap water which was the common source to all pools. The lowest chronic cytotoxicity was expressed by the organics isolated from tap water (PS3) with a LC50 concentration factor of 346. The higher cytotoxicity of the recreational pool waters compared to the tap water source may reflect prolonged disinfectant contact times. Tap water disinfectant contact times are on the order of 1 week, whereas in recreational pools residence times are on the order of months. Another factor for the elevated cytotoxicity in the pool waters may be due to different organic matter precursors contributed by bathers. Compared to the carbon-rich humic acid precursors in many drinking water source waters, organics contributed by urine, sweat, hair, skin, and consumer products contain nitrogen-rich precursors. Elevated genotoxicity and cytotoxicity are associated with many classes of N-DBPs.17,19 Indeed, all of the pools exhibited TOC concentrations greater than the 1.2 mg/L measured in the source tap water. The TOC of most pools ranged from 5.2 to 33.1 mg/L, reflecting the contribution of organic precursors from bathers. However, the TOC for pool PS4 was 124.8 mg/L, likely due to the accumulation of the organic BCDMH disinfectant and its organic degradation products. Important differences were observed based upon disinfectant type and environmental conditions. Illumination conditions affected the cytotoxicity of the water. Samples were taken from a single recreational pool that was enclosed during cold weather (PS8) and opened during warm weather (PS9). The pool water under indoor conditions was more cytotoxic (LC50 = 24.2 ) than when it was operated as an outdoor pool (LC50 = 181.4 ) (Table 2). Although the TOC and total chlorine residual were similar for PS8 and PS9, the cytotoxic potency of the pool was nearly 8 lower with solar exposure and perhaps enhanced volatilization. Ultraviolet light combined with free chlorine disinfection of indoor pools appeared to reduce the cytotoxicity of the water. Despite having comparable levels of TOC and total chlorine residual, the cytotoxicity of the pools disinfected with free chlorine alone (PS5 and PS8) was approximately 3 higher 4162

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Figure 2. Comparison of the CHO cell cytotoxicity index values and the genotoxicity index values35 for the water samples evaluated in this study. Index values are expressed in arbitrary units. The association between cytotoxicity and genotoxicity is directly and significantly correlated (r = 0.74; P e 0.03).

than the pool disinfected with free chlorine and UV (PS1 and PS7). Solar and UV radiation may decompose DBPs. For example one study demonstrated that nitrosamine concentrations were reduced approximately 5 in outdoor versus indoor pools.22 Among indoor pools at room temperature, the pool (PS4) disinfected with BCDMH exhibited cytotoxicity comparable (LC50 = 21.9 ) to those disinfected with free chlorine (LC50 for PS5 = 20.3 and LC50 for PS8 = 24.2 ), even though pool PS4 exhibited a TOC at least four times higher than all other pools. As BCDMH is organic, the high TOC likely reflects the accumulation of either BCDMH or its organic degradation products in the pool, rather than elevated precursors from bathers and their associated byproducts. Other environmental conditions appeared less relevant. A Pearson’s Product Moment Correlation analysis between TOC and cytotoxic potency failed to resolve a significant association (r = 0.37; P = 0.33). Although samples PS1 and PS7, collected at different times from the same pool, exhibited similar cytotoxicity, sample PS1 had TOC and total residual chlorine concentrations more than 2 higher than sample PS7. Samples collected from a cold (PS5), or warm pool (PS6), or a hot tub (PS2) at the same facility showed no significant correlation between cytotoxicity and temperature (r =
0.32; P = 0.79). A direct significant association was observed between cytotoxicity (this study) and genomic DNA damage in CHO cells,35 (Figure 2) (r = 0.74; P e 0.03). Although there was a clear association between in vitro chronic cytotoxic potency and acute genotoxic potency, there was a notable difference in magnitude of response with PS4 (a pool treated with BCDMH). Sample PS4 expressed a high level of genotoxicity (Figure 2) and we hypothesize that this is due to the enhanced generation of brominated and nitrogenous-DBPs (N-DBPs). Previous studies demonstrated that brominated and N-DBPs were more genotoxic than their chlorinated analogues.17,19 These data indicate that brominating agents should be avoided as disinfectants of recreational pool water.

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Our study represents the first systematic mammalian cell cytotoxicity analysis to evaluate different recreational waters derived from a common tap water source. All disinfected recreational pool water samples were more cytotoxic than the source tap water. Of primary importance in limiting the cytotoxicity of the pool waters were the illumination conditions. The indoor pool treated with a combination of ultraviolet and free chlorine exhibited lower cytotoxicity than comparable pools disinfected with free chlorine alone. The outdoor pool exposed to sunlight featured lower cytotoxicity than the same pool under indoor conditions. Cytotoxicity correlated with genotoxicity. The results indicated that either the compounds responsible for the cytotoxicity, or their precursors, may be photolabile. Our results indicate that combining ultraviolet treatment with free chlorine may be effective for reducing the bulk toxicity of recreational waters, a finding that should be validated in additional pools. Accordingly, care should be taken in the disinfectant employed to treat recreational pool water. A reduction in TOC may reduce the level of toxic byproducts in pool waters; this may be achieved by reducing cosmetics and personal care products, some of which are converted into toxic agents after chlorination.41 During recycling of pool water, the organic carbon could be removed by materials such as granulated activated carbon. Of importance in reducing the precursors of toxic DBPs, swimmers should be encouraged to improve their hygienic behavior by showering before entering the water and patrons should be informed about the potential harm from urinating in a pool. A common practice in epidemiological studies uses trihalomethanes to monitor exposure. Because the concentrations of trihalomethanes are similar between tap waters and pool waters,42
44 the results of our study suggest that trihalomethanes may not provide sufficient resolving power. The identity of specific toxic agents responsible for cytotoxicity and genotoxicity is unclear. Cytotoxicity is a sensitive metric for adverse biological responses, and it may be a more appropriate approach to monitor the possible health hazards associated with disinfected pool water. Several in vitro cytotoxicity end points are being used by the NIH Chemical Genomics Center and their quantitative high throughput screening (qHTS) technologies.45 For water samples cytotoxicity may be a highly useful measure for nongenotoxic (and perhaps noncarcinogenic) health end points such as skin and respiratory ailments,14,46 asthma in children,7,9,10 and risks to pregnant women.43,47
49 Short-term, in vitro cytotoxicity measurements may be valuable indicators to monitor recreational waters and evaluate the impact of alternative disinfectants or modified behavior of bathers.

’ AUTHOR INFORMATION Corresponding Author

*Telephone: 217-333-3614; e-mail: [email protected].

’ ACKNOWLEDGMENT This research was supported by grants from the National Science Foundation (CBET-0651732 and CBET-0651333). We appreciate the support by the Center of Advanced Materials for the Purification of Water with Systems, National Science Foundation Science and Technology Center, under Award CTS0120978. 4163

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