Article pubs.acs.org/est
Integrated Chemical and Toxicological Investigation of UV-Chlorine/ Chloramine Drinking Water Treatment Bonnie A. Lyon,†,# Rebecca Y. Milsk,† Anthony B. DeAngelo,§ Jane Ellen Simmons,§ Mary P. Moyer,∥ and Howard S. Weinberg*,† †
Department of Environmental Sciences and Engineering, Gillings School of Global Public Health, University of North Carolina at Chapel Hill, 146A Rosenau Hall, Chapel Hill, North Carolina 27599, United States § National Health & Environmental Effects Research Laboratory, Office of Research and Development, United States Environmental Protection Agency, 109 T.W. Alexander Drive, Research Triangle Park, North Carolina 27711, United States ∥ INCELL Corporation, 12734 Cimarron Path, San Antonio, Texas 78249, United States S Supporting Information *
ABSTRACT: As the use of alternative drinking water treatment increases, it is important to understand potential public health implications associated with these processes. The objective of this study was to evaluate the formation of disinfection byproducts (DBPs) and cytotoxicity of natural organic matter (NOM) concentrates treated with chlorine, chloramine, and medium pressure ultraviolet (UV) irradiation followed by chlorine or chloramine, with and without nitrate or iodide spiking. The use of concentrated NOM conserved volatile DBPs and allowed for direct analysis of the treated water. Treatment with UV prior to chlorine in ambient (unspiked) samples did not affect cytotoxicity as measured using an in vitro normal human colon cell (NCM460) assay, compared to chlorination alone when toxicity is expressed on the basis of dissolved organic carbon (DOC). Nitrate-spiked UV+chlorine treatment produced greater cytotoxicity than nitrate-spiked chlorine alone or ambient UV+chlorine samples, on both a DOC and total organic halogen basis. Samples treated with UV +chloramine were more cytotoxic than those treated with only chloramine using either dose metric. This study demonstrated the combination of cytotoxicity and DBP measurements for process evaluation in drinking water treatment. The results highlight the importance of dose metric when considering the relative toxicity of complex DBP mixtures formed under different disinfection scenarios.
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INTRODUCTION Drinking water disinfection was one of the greatest public health advances of the 20th century, and it vastly reduced cholera and typhoid outbreaks.1 In the mid-1970s, byproducts of the chlorination process were discovered, including chloroform and other trihalomethanes (THMs).2,3 Disinfection byproducts (DBPs) are formed when a disinfectant reacts with ubiquitous decaying plant and microbial matter (natural organic matter, NOM), salts in water that can be of natural origin (e.g., bromide, iodide), or anthropogenic pollutants. Shortly after the discovery of DBPs in drinking water, a report by the National Cancer Institute showed that chloroform was carcinogenic in laboratory animals,4 and another study demonstrated that concentrated organic extracts of chlorinated waters were mutagenic.5 The United States Environmental Protection Agency currently regulates a subset of two major DBP classes, chosen as indicators for overall DBP formation: four chlorine- and bromine-containing THMs (THM4) and five chlorine- and bromine-containing haloacetic acids (HAA5), along with bromate and chlorite.6 Studies in rodents have © 2014 American Chemical Society
shown that nearly all of the 20 DBPs evaluated for carcinogenicity and approximately 100 DBPs assessed in vitro for genotoxicity/mutagenicity test positive.7 Epidemiological studies suggest that chlorination byproducts may increase the risk of bladder and colon cancer8−12 as well as adverse reproductive and developmental effects,13−16 emphasizing the importance of balancing microbial inactivation and chemical byproduct risk. However, inconsistent findings among epidemiological studies have yielded uncertainty regarding these associations.17 Single-compound, in vitro toxicological assays have shown that nitrogen-containing DBPs are more geno- and cytotoxic than the regulated THM4 and HAA5 species and that bromineand iodine-substituted DBPs are more toxic than their corresponding chlorine-containing byproducts.7,18−22 CombinReceived: Revised: Accepted: Published: 6743
March 23, 2014 May 13, 2014 May 19, 2014 May 19, 2014 dx.doi.org/10.1021/es501412n | Environ. Sci. Technol. 2014, 48, 6743−6753
Environmental Science & Technology
Article
ing in vitro toxicity data with DBP occurrence information can provide insight into ranking DBPs or DBP classes for further consideration. Although more than 600 individual DBPs have been identified in laboratory and field studies resulting from a variety of treatments and disinfectants, there remains a large percentage of unidentified byproducts as indicated by analysis of total organic halogen.23,24 A measure of total organic halogen can be compared to the chlorine equivalents of individually measured halogen-containing DBPs to determine the percentage of unidentified halogenated organics in disinfected water. Little is known about what is contained within this unknown fraction and how it contributes to the total toxicity of treated water. Bull and colleagues reported that epidemiological findings associated with disinfected drinking water could not be accounted for by the regulated DBPs alone.25 Thus, there is a need for research focused on identifying the potential health effects of DBP mixtures present in treated water samples, which include known and unknown byproducts and are more representative of what consumers are actually exposed to on a regular basis. As a result of anthropogenic impacts on source water quality and to meet DBP regulations, utilities have been looking at alternative treatment processes to chlorination (e.g., chloramines, ozone, chlorine dioxide). An occurrence study of treated drinking waters across the United States found that while the use of alternative disinfectants reduced regulated THM4 and HAA5 formation, in some cases, these processes also increased the formation of DBPs thought to be more genoand cytotoxic than their regulated counterparts.24 Ultraviolet (UV) irradiation is an alternative treatment process that is being increasingly used for drinking water, and it is very effective at inactivating chlorine-resistant pathogens such as Cryptosporidium.26 For treatment of surface waters in North America, UV is used in combination with a secondary disinfectant such as chlorine or chloramine to provide a distribution residual. Low pressure (LP, monochromatic output at 253.7 nm) and medium pressure (MP, polychromatic output 200−400 nm) are the two commonly used lamp types for UV treatment of drinking water. Several studies have investigated DBP formation from UV-chlorine/chloramine treatment27−30 focusing mostly on the regulated THM4 and HAA5, although in two studies, disinfection doses of MP UV (40−186 mJ/cm2) in combination with chlorine or chloramine were shown to increase the formation of unregulated halonitromethanes in waters containing 1−10 mg N/L nitrate.31,32 Another study found that LP and MP UV treatment followed by chlorine/ chloramine increased cyanogen chloride formation by up to 134% in Suwannee River NOM isolates and a real drinking water source.29 In addition to DBP analysis, toxicological studies are important for evaluating alternative treatment processes. Although the necessity of in vivo animal studies for estimating human health risk to DBPs through multiple exposure routes and different end points is recognized, such experiments are more resource intensive with lower throughput than in vitro assays. In vitro assays may not be able to directly predict human health consequences, but they can serve as a powerful tool for comparative toxicology. Another advantage of in vitro experiments is that they can be carried out using human cells. Aside from a limited number of in vivo and in vitro complex DBP mixture studies,33−39 the majority of past DBP toxicology work has tested single compounds or simple, defined mixtures that are not representative of a real disinfected drinking water.
This study aimed to evaluate DBP formation in the context of potential human health implications. Accordingly, the objective of this work was to use an in vitro chronic cytotoxicity assay in combination with DBP and total organic halogen analysis to determine the formation and cytotoxicity of byproducts in water produced during MP UV-chlorine/ chloramine treatment. Most toxicological assays require sample concentration to induce a measurable response. Therefore, an approach in which preconcentrated NOM40 was treated and then applied directly to the assay without any further concentration was used in the current work. Additional advantages of this approach are that the sample remained in an aqueous matrix and minimal headspace or headspace-free conditions were maintained to minimize loss of volatile DBPs. The assay used normal human colon cells, a relevant target cell for investigating human health effects from DBPs because of the association of chlorinated water consumption with colorectal cancer.41 The results were compared to the same waters treated with only chlorine or chloramine at a dose adjusted to provide a similar residual. A subset of samples was spiked with nitrate or iodide to investigate the impact of these inorganic precursors on byproduct formation and cytotoxicity in the treated waters.
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MATERIALS AND METHODS Sample Preparation. Nordic Reservoir NOM was obtained as a low-ash freeze-dried isolate from the International Humic Substances Society (St. Paul, MN, USA). It was determined through preliminary tests that a dissolved organic carbon (DOC) concentration of 125 mg C/L generated enough DBPs upon chlorination/chloramination to induce a measurable response in the cytotoxicity assay while lowering the cell density to approximately 82% of the control cells (Supporting Information (SI) Figure S1). To reduce this latter effect, a concentration of 112 mg C/L, which was subsequently diluted to 90 mg C/L during the cytotoxicity assay sample preparation procedure, was used in disinfection experiments where cell density was approximately 88%. All samples were prepared at this DOC concentration so that any background effects were consistent and comparisons could be made across treatments. To prepare an aqueous solution of Nordic Reservoir NOM, the solid NOM isolate was weighed out, dissolved in laboratory grade water (LGW), filtered (0.45 μm), and stored in an amber glass bottle at 4 °C until use. The SUVA254 value of this water was 4.5 L/mg-C m, which is higher than typical raw source waters. In comparison, the median SUVA254 for raw waters at 12 U.S. drinking water treatment plants was 2.9 L/mg-C m.42 Additional characteristics are shown in the SI Table S1. LGW was prepared in-house from a Dracor system (Durham, NC, USA), which prefilters inlet 7 MΩ deionized water to 1 μm, removes residual disinfectants, reduces total organic carbon (TOC) to less than 0.2 mg C/L with an activated carbon resin, and removes ions to 18 MΩ with mixed bed ion-exchange resins. DOC and total dissolved nitrogen (TDN) were measured using a Shimadzu TOC-VCPH Total Organic Carbon Analyzer with a TNM-1 Total Nitrogen Measuring Unit (Shimadzu Corporation, Atlanta, GA, USA) following Standard Method 5310.43 Prior to chlorination/chloramination, samples were buffered with 20 mM phosphate buffer at pH 7.1 (pH for target cells). Ambient (unspiked) samples contained 0.2 mg N/L nitrate and
Integrated Chemical and Toxicological Investigation of UV-Chlorine/ Chloramine Drinking Water Treatment Bonnie A. Lyon,†,# Rebecca Y. Milsk,† Anthony B. DeAngelo,§ Jane Ellen Simmons,§ Mary P. Moyer,∥ and Howard S. Weinberg*,† †
Department of Environmental Sciences and Engineering, Gillings School of Global Public Health, University of North Carolina at Chapel Hill, 146A Rosenau Hall, Chapel Hill, North Carolina 27599, United States § National Health & Environmental Effects Research Laboratory, Office of Research and Development, United States Environmental Protection Agency, 109 T.W. Alexander Drive, Research Triangle Park, North Carolina 27711, United States ∥ INCELL Corporation, 12734 Cimarron Path, San Antonio, Texas 78249, United States S Supporting Information *
ABSTRACT: As the use of alternative drinking water treatment increases, it is important to understand potential public health implications associated with these processes. The objective of this study was to evaluate the formation of disinfection byproducts (DBPs) and cytotoxicity of natural organic matter (NOM) concentrates treated with chlorine, chloramine, and medium pressure ultraviolet (UV) irradiation followed by chlorine or chloramine, with and without nitrate or iodide spiking. The use of concentrated NOM conserved volatile DBPs and allowed for direct analysis of the treated water. Treatment with UV prior to chlorine in ambient (unspiked) samples did not affect cytotoxicity as measured using an in vitro normal human colon cell (NCM460) assay, compared to chlorination alone when toxicity is expressed on the basis of dissolved organic carbon (DOC). Nitrate-spiked UV+chlorine treatment produced greater cytotoxicity than nitrate-spiked chlorine alone or ambient UV+chlorine samples, on both a DOC and total organic halogen basis. Samples treated with UV +chloramine were more cytotoxic than those treated with only chloramine using either dose metric. This study demonstrated the combination of cytotoxicity and DBP measurements for process evaluation in drinking water treatment. The results highlight the importance of dose metric when considering the relative toxicity of complex DBP mixtures formed under different disinfection scenarios.
■
INTRODUCTION Drinking water disinfection was one of the greatest public health advances of the 20th century, and it vastly reduced cholera and typhoid outbreaks.1 In the mid-1970s, byproducts of the chlorination process were discovered, including chloroform and other trihalomethanes (THMs).2,3 Disinfection byproducts (DBPs) are formed when a disinfectant reacts with ubiquitous decaying plant and microbial matter (natural organic matter, NOM), salts in water that can be of natural origin (e.g., bromide, iodide), or anthropogenic pollutants. Shortly after the discovery of DBPs in drinking water, a report by the National Cancer Institute showed that chloroform was carcinogenic in laboratory animals,4 and another study demonstrated that concentrated organic extracts of chlorinated waters were mutagenic.5 The United States Environmental Protection Agency currently regulates a subset of two major DBP classes, chosen as indicators for overall DBP formation: four chlorine- and bromine-containing THMs (THM4) and five chlorine- and bromine-containing haloacetic acids (HAA5), along with bromate and chlorite.6 Studies in rodents have © 2014 American Chemical Society
shown that nearly all of the 20 DBPs evaluated for carcinogenicity and approximately 100 DBPs assessed in vitro for genotoxicity/mutagenicity test positive.7 Epidemiological studies suggest that chlorination byproducts may increase the risk of bladder and colon cancer8−12 as well as adverse reproductive and developmental effects,13−16 emphasizing the importance of balancing microbial inactivation and chemical byproduct risk. However, inconsistent findings among epidemiological studies have yielded uncertainty regarding these associations.17 Single-compound, in vitro toxicological assays have shown that nitrogen-containing DBPs are more geno- and cytotoxic than the regulated THM4 and HAA5 species and that bromineand iodine-substituted DBPs are more toxic than their corresponding chlorine-containing byproducts.7,18−22 CombinReceived: Revised: Accepted: Published: 6743
March 23, 2014 May 13, 2014 May 19, 2014 May 19, 2014 dx.doi.org/10.1021/es501412n | Environ. Sci. Technol. 2014, 48, 6743−6753
Environmental Science & Technology
Article
ing in vitro toxicity data with DBP occurrence information can provide insight into ranking DBPs or DBP classes for further consideration. Although more than 600 individual DBPs have been identified in laboratory and field studies resulting from a variety of treatments and disinfectants, there remains a large percentage of unidentified byproducts as indicated by analysis of total organic halogen.23,24 A measure of total organic halogen can be compared to the chlorine equivalents of individually measured halogen-containing DBPs to determine the percentage of unidentified halogenated organics in disinfected water. Little is known about what is contained within this unknown fraction and how it contributes to the total toxicity of treated water. Bull and colleagues reported that epidemiological findings associated with disinfected drinking water could not be accounted for by the regulated DBPs alone.25 Thus, there is a need for research focused on identifying the potential health effects of DBP mixtures present in treated water samples, which include known and unknown byproducts and are more representative of what consumers are actually exposed to on a regular basis. As a result of anthropogenic impacts on source water quality and to meet DBP regulations, utilities have been looking at alternative treatment processes to chlorination (e.g., chloramines, ozone, chlorine dioxide). An occurrence study of treated drinking waters across the United States found that while the use of alternative disinfectants reduced regulated THM4 and HAA5 formation, in some cases, these processes also increased the formation of DBPs thought to be more genoand cytotoxic than their regulated counterparts.24 Ultraviolet (UV) irradiation is an alternative treatment process that is being increasingly used for drinking water, and it is very effective at inactivating chlorine-resistant pathogens such as Cryptosporidium.26 For treatment of surface waters in North America, UV is used in combination with a secondary disinfectant such as chlorine or chloramine to provide a distribution residual. Low pressure (LP, monochromatic output at 253.7 nm) and medium pressure (MP, polychromatic output 200−400 nm) are the two commonly used lamp types for UV treatment of drinking water. Several studies have investigated DBP formation from UV-chlorine/chloramine treatment27−30 focusing mostly on the regulated THM4 and HAA5, although in two studies, disinfection doses of MP UV (40−186 mJ/cm2) in combination with chlorine or chloramine were shown to increase the formation of unregulated halonitromethanes in waters containing 1−10 mg N/L nitrate.31,32 Another study found that LP and MP UV treatment followed by chlorine/ chloramine increased cyanogen chloride formation by up to 134% in Suwannee River NOM isolates and a real drinking water source.29 In addition to DBP analysis, toxicological studies are important for evaluating alternative treatment processes. Although the necessity of in vivo animal studies for estimating human health risk to DBPs through multiple exposure routes and different end points is recognized, such experiments are more resource intensive with lower throughput than in vitro assays. In vitro assays may not be able to directly predict human health consequences, but they can serve as a powerful tool for comparative toxicology. Another advantage of in vitro experiments is that they can be carried out using human cells. Aside from a limited number of in vivo and in vitro complex DBP mixture studies,33−39 the majority of past DBP toxicology work has tested single compounds or simple, defined mixtures that are not representative of a real disinfected drinking water.
This study aimed to evaluate DBP formation in the context of potential human health implications. Accordingly, the objective of this work was to use an in vitro chronic cytotoxicity assay in combination with DBP and total organic halogen analysis to determine the formation and cytotoxicity of byproducts in water produced during MP UV-chlorine/ chloramine treatment. Most toxicological assays require sample concentration to induce a measurable response. Therefore, an approach in which preconcentrated NOM40 was treated and then applied directly to the assay without any further concentration was used in the current work. Additional advantages of this approach are that the sample remained in an aqueous matrix and minimal headspace or headspace-free conditions were maintained to minimize loss of volatile DBPs. The assay used normal human colon cells, a relevant target cell for investigating human health effects from DBPs because of the association of chlorinated water consumption with colorectal cancer.41 The results were compared to the same waters treated with only chlorine or chloramine at a dose adjusted to provide a similar residual. A subset of samples was spiked with nitrate or iodide to investigate the impact of these inorganic precursors on byproduct formation and cytotoxicity in the treated waters.
■
MATERIALS AND METHODS Sample Preparation. Nordic Reservoir NOM was obtained as a low-ash freeze-dried isolate from the International Humic Substances Society (St. Paul, MN, USA). It was determined through preliminary tests that a dissolved organic carbon (DOC) concentration of 125 mg C/L generated enough DBPs upon chlorination/chloramination to induce a measurable response in the cytotoxicity assay while lowering the cell density to approximately 82% of the control cells (Supporting Information (SI) Figure S1). To reduce this latter effect, a concentration of 112 mg C/L, which was subsequently diluted to 90 mg C/L during the cytotoxicity assay sample preparation procedure, was used in disinfection experiments where cell density was approximately 88%. All samples were prepared at this DOC concentration so that any background effects were consistent and comparisons could be made across treatments. To prepare an aqueous solution of Nordic Reservoir NOM, the solid NOM isolate was weighed out, dissolved in laboratory grade water (LGW), filtered (0.45 μm), and stored in an amber glass bottle at 4 °C until use. The SUVA254 value of this water was 4.5 L/mg-C m, which is higher than typical raw source waters. In comparison, the median SUVA254 for raw waters at 12 U.S. drinking water treatment plants was 2.9 L/mg-C m.42 Additional characteristics are shown in the SI Table S1. LGW was prepared in-house from a Dracor system (Durham, NC, USA), which prefilters inlet 7 MΩ deionized water to 1 μm, removes residual disinfectants, reduces total organic carbon (TOC) to less than 0.2 mg C/L with an activated carbon resin, and removes ions to 18 MΩ with mixed bed ion-exchange resins. DOC and total dissolved nitrogen (TDN) were measured using a Shimadzu TOC-VCPH Total Organic Carbon Analyzer with a TNM-1 Total Nitrogen Measuring Unit (Shimadzu Corporation, Atlanta, GA, USA) following Standard Method 5310.43 Prior to chlorination/chloramination, samples were buffered with 20 mM phosphate buffer at pH 7.1 (pH for target cells). Ambient (unspiked) samples contained 0.2 mg N/L nitrate and
