Comparative in Vitro Toxicity of Nitrosamines and Nitramines

Overall, N-nitrosamines were ∼15-fold more mutagenic than their N-nitramine ... (1) One approach to reduce CO2 levels involves carbon capture and st...
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Comparative in Vitro Toxicity of Nitrosamines and Nitramines Associated with Amine-based Carbon Capture and Storage Elizabeth D. Wagner,†,‡ Jennifer Osiol,† William A. Mitch,§ and Michael J. Plewa*,†,‡ †

Department of Crop Sciences, University of Illinois at Urbana−Champaign, Urbana, Illinois 61801, United States Center for Advanced Materials for the Purification of Water with Systems, and the Global Safe Water Institute, University of Illinois at Urbana−Champaign, Urbana, Illinois 61801, United States § Department of Civil and Environmental Engineering, Stanford University, Stanford, California 94305, United States ‡

S Supporting Information *

ABSTRACT: Amine-based CO2 capture is a prime contender for the first full-scale implementation of CO2 capture at fossil fuel-fired power plants postcombustion. However, the formation of potentially carcinogenic N-nitrosamines and N-nitramines from reactions of flue gas NOx with the amines presents a potential risk for contaminating airsheds and drinking water supplies. Setting regulatory emission limits is hampered by the dearth of toxicity information for the N-nitramines. This study employed quantitative in vitro bioassays for mutagenicity in Salmonella typhimurium, and chronic cytotoxicity and acute genotoxicity in Chinese hamster ovary (CHO) cells to compare the toxicity of analogous N-nitrosamines and N-nitramines relevant to CO2 capture. Although the rank order was similar for genotoxicity in CHO cells and mutagenicity in S. typhimurium, the Salmonella assay was far more sensitive. In general, mutagenicity was higher with S9 hepatic microsomal activation. The rank order of mutagenicity was N-nitrosodimethylamine (NDMA) > N-nitrosomorpholine > N-nitrodimethylamine >1,4-dinitrosopiperazine > N-nitromorpholine >1,4-dinitropiperazine > N-nitromonoethanolamine > N-nitrosodiethanolamine > N-nitrodiethanolamine. 1-Nitrosopiperazine and 1-nitropiperazine were not mutagenic. Overall, N-nitrosamines were ∼15-fold more mutagenic than their N-nitramine analogues.

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INTRODUCTION Climate change through the release of CO2 from fossil fuel combustion is a recognized global crisis.1 One approach to reduce CO2 levels involves carbon capture and storage (CCS). CCS technologies enable the capture of CO2 from fossil fuel combustion and its ultimate storage in appropriate geological formations. The International Energy Agency estimates that CCS will account for approximately 20% of the CO2 reductions needed by 2050. To reach this goal, approximately 100 largescale CCS installations are required by 2020 with over 3000 by 2050.2 Large-scale postcombustion CO2 sequestration will play a critical role in the global transition to a sustainable low-carbon economy in both power generation and industry.3 With authorization from the Norwegian government, Gassnova and partners planned the installation of one of the first large-scale CO2 capture plants at a combined heat and power plant at Mongstad, Norway; this plant would have sequestered up to 1.2 × 106 metric tons of CO2 per year,4 although the Norwegian Government canceled the project in September 2013. Norway has been at the forefront of carbon capture. Since 1996, Statoil used amine-based technologies to capture CO2 from North Sea natural gas streams precombustion with the captured CO2 stored in North Sea geological formations. For amine-based capture systems, power plant flue gases pass through an absorber column at ∼50 °C with a counter-current amine-based aqueous solvent (e.g., 30% monoethanolamine).5 After © 2014 American Chemical Society

dissolution of the CO2 into the solvent, the CO2 forms complexes with the amines. Tertiary amines and sterically hindered primary and secondary amines form bicarbonate complexes (eq 1 for methyl diethanolamine), whereas unhindered primary and secondary amines form carbamate complexes (eq 2 for monoethanolamine). The solvent is then routed to a desorber column where high temperatures (∼100−150 °C) reverse the CO2 complex formation reactions (eqs 1 and 2) to release CO2. The CO2 is compressed and routed to geological storage while the amines are sent back to the absorber column. The flue gas exiting the absorber column is generally routed through a counter-current washwater column to scrub amines and other potential contaminants prior to atmospheric release.

Amine-based technologies are favored for the first full-scale attempts at postcombustion CO2 capture, because these Received: Revised: Accepted: Published: 8203

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technologies can be incorporated onto the effluent of existing power plants, and because there have been nearly two decades of full-scale experience with these technologies for removal of CO2 from natural gas.6,7 However, a significant difference between precombustion and postcombustion applications is the presence of NOx in the flue gases entering the capture units for postcombustion applications. NOx constituents NO and NO2 can form N2O3 (eq 3). Reaction of N2O3 with amines forms nitrosamines (eq 5 for piperazine). Dimerization of NO2 can form N2O4 (eq 4), which has two tautomeric forms, ONONO2 and O2NNO2.8 ONONO2 is believed to react with amines to form nitrosamines (eq 6), whereas reaction of O2NNO2 with amines forms nitramines (eq 7). Nitrosamine and nitramine formation could occur within the absorber column via these reactions.9,10 However, nitrite forms in the solvent both by eqs 5 and 7, as well as by hydrolysis of N2O3 and N2O4 (eqs 8 and 9). Under the high temperature conditions prevailing within desorber units, nitrite is considered to be a potent nitrosating agent.10,11 Nitrosamines and nitramines accumulate within the washwater due to volatilization from the absorber unit. Although intended to scrub contaminants, including nitrosamines and nitramines, washwater units may also serve as a source of nitrosamines and nitramines due to reactions of residual NOx with the amines that accumulate with the washwater.12 Accordingly, concerns for postcombustion implementation of amine-based CO2 capture systems center around the potential for contamination of downwind airsheds and drinking water aquifers by nitrosamines and nitramines that may volatilize from washwater units or that may form from the reactions of amines volatilizing from washwater units with ambient NOx in the atmosphere.5,7 Initial measurements of total nitrosamine concentrations in pilot plant washwater samples indicated concentrations of 0.50−0.73 μM for monoethanolamine-based solvents and 59 μM for a solvent consisting of 25% 2-amino-2-methyl-1-propanol and 15% piperazine.13 These results concur with a study that indicated that solvents containing secondary amines produce nitrosamines at roughly 2 orders of magnitude higher levels than solvents based on primary amines.11 Nitrosamine formation generally far exceeded nitramine formation;9 even in the presence of 25 ppm by volume (ppmv) NO2 and 0 ppmv NO, a condition that should favor formation of the potential nitrating agent, N2O4 (eqs 4, 5, and 7), nitrosamine formation exceeded nitramine formation by a factor of 3.13 However, although nitrosamines may be subject to sunlight photolysis,14−17 nitramines do not absorb sunlight. Thus, nitramines may be of particular concern because if they are released from CO2 capture plants or if they form within the atmosphere via reactions of amines with ambient NOx, they may be less likely to degrade prior to contaminating airsheds or drinking water sources. NO + NO2 ⇌ N2O3

(3)

2NO2 ⇌ N2O4

(4)

Specific nitrosamines are potent carcinogens and have been studied for their toxicity as components of tobacco smoke and cured meats.18,19 There are concerns for the occurrence of nitrosamines in drinking waters, where they form as byproducts of chloramine disinfection.20−24 The U.S. EPA Integrated Risk Information System (IRIS) database indicates for several nitrosamines that low ng/L concentrations in drinking water are associated with a 10−6 excess lifetime cancer risk.25 California has established 10 ng/L notification levels for N-nitrosodimethylamine (NDMA), N-nitrosodiethylamine (NDEA), and N-nitrosodi-N-propylamine (NDPA).26 The U.S. EPA is considering nationwide regulations for nitrosamines in drinking waters. Exceedingly limited comparative toxicity information for nitramines exists. Although nitramines form as byproducts of disinfection in recreational waters,27 they tend not to form as byproducts of chloramine disinfection of drinking waters. Limited data are available for animal toxicity and carcinogenicity for N-nitrodimethylamine;28−30 this agent was identified as a promutagen.31−34 Recently, Dusinska and colleagues analyzed CCS-associated nitramines (N-nitrodimethylamine, N-nitromethylamine, N-nitroethanolamine, and 2-methyl-2-(nitroamino)-1-propanol) with the Bovine Corneal Opacity and Permeability assay; although none of the four nitramines were skin irritants, they expressed varied levels of eye irritation response.35 Whether nitramines exhibit cancer potencies comparable to those of nitrosamines represents a key question for the development of regulatory levels for aminebased CO2. In the absence of this information, the Norwegian Climate and Pollution Agency considered the cancer potencies to be equivalent, issuing a permit for the CO2 capture facility at Mongstad, 36 Norway that limited the total concentrations of N-nitrosamines and N-nitramines in airsheds and water supplies downwind of the plant to 0.3 ng/m3 and 4 ng/L, respectively.37 The goal of this research was to quantitatively evaluate the mutagenicity and genotoxicity of nitramines associated with CCS technologies. The specific objectives of this study were (1) to analyze the mutagenicity in Salmonella typhimurium of six nitramines (N-nitrodiethanolamine, N-nitromonoethanolamine, N-nitromorpholine, N-nitrodimethylamine, 1-nitropiperazine, and 1,4-dinitropiperazine) and their nitrosamine analogues, (2) to determine the induction of chronic cytotoxicity and acute genomic DNA damage of these nitramines and nitrosamines in mammalian cells, and (3) to compare the genotoxic potencies of these nitramines with that of their nitrosamine 8204

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Figure 1. Cytotoxicity and mutagenicity concentration−response curves for nitrosamines and nitramines with S. typhimurium YG7108 using a microplate suspension assay. Top panels (A, C, E, G, I, K) show the cytotoxicity of the chemicals −S9 (filled symbols) or +S9 (open symbols). Lower panels (B, D, F, H, J, L) show the mutagenicity of the chemicals, −S9 (filled symbols) or +S9 (open symbols).

concentration of 0.7 ng/L NDMA is associated with a 10−6 lifetime excess cancer risk.25 A final goal is to develop an equivalency metric of these agents to N-nitrosodimethylamine for future risk assessment.

analogues. Finally, N-nitrosodimethylamine (NDMA) has been a focus of recent drinking water research due to its formation as a byproduct of chloramine disinfection.20,22 The U.S. EPA IRIS database indicates that a drinking water 8205

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Table 1. Comparative Mutagenicity of Nitrosamines and Nitramines in S. typhimurium Salmonella typhimurium YG7108 Reversion Assay Compound

conc. range (μM)

S9a

2× mutat. conc. (μM)b

r2c

mutagenic potency (rev/μmol)d

N-nitrosodimethylamine (NDMA) N-nitrosodimethylamine (NDMA) N-nitrodimethylamine (NO2DMA) N-nitrodimethylamine (NO2DMA) N-nitrosomorpholine (NMOR) N-Nitrosomorpholine (NMOR) N-nitromorpholine (NO2MOR) N-nitromorpholine (NO2MOR) N-nitrosodiethanolamine (NDELA) N-nitrosodiethanolamine (NDELA) N-Nitrodiethanolamine (NO2DELA) N-nitrodiethanolamine (NO2DELA) 1-nitrosopiperazine (NPZ) 1-nitrosopiperazine (NPZ) 1-nitropiperazine (NO2PZ) 1-nitropiperazine (NO2PZ) 1,4-dinitrosopiperazine (DNPZ) 1,4-dinitrosopiperazine (DNPZ) 1,4-dinitropiperazine (DNO2PZ) 1,4-dinitropiperazine (DNO2PZ) N-nitromonoethanolamine N-nitromonoethanolamine

50−5000 1.25−10000 50−7500 50−7500 100−7500 5−7500 100−7500 100−7500 7500−30000 7500−30000 10000−30000 7500−30000 100−7500 100−7500 500−10000 500−7500 100−7500 100−7500 5000−20000 1000−20000 5000−30000 5000−30000

−S9 +S9 −S9 +S9 −S9 +S9 −S9 +S9 −S9 +S9 −S9 +S9 −S9 +S9 −S9 +S9 −S9 +S9 −S9 +S9 −S9 +S9

negative 4.5 negative 279.6 negative 21.4 negative 1177 26838 10304 25193 negative negative negative negative negative negative 49.1* 2118* 1099 1732* 1366*

NA 0.98 NA 0.99 NA 0.99 NA 0.99 0.95 0.95 0.95 NA NA NA NA NA NA 0.99 0.99 0.99 0.99 0.99

negative 21642 negative 1660 negative 6724 negative 272 10 22 8 negative negative negative negative negative negative 1263 157 191 134 133

a

Aroclor-induced hepatic microsomal mixture (MolTox Inc.). bThe concentration of the test compound that would induce a 2× increase over the number of spontaneous revertants per VB plate (as derived from the regression analysis). Lower concentrations denote greater mutagenicity. The values designated with an asterisk were calculated by extrapolating concentration−response curves employed in the experiments. cCoefficient of determination of the goodness of fit for the regression analyses used for the 2× concentration value and for the mutagenic potency. dThe mutagenic potency is the average induced revertants per μmol calculated from the concentration−response curve. Larger mutagenic potency values denote greater mutagenicity. NA = not applicable.

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O6-methylguanine DNA methyltransferases involved in the repair of DNA damage caused by alkylating agents. We chose strain YG7108 because of its sensitivity to alkylating agents including N-nitrosodimethylamine.38 Further information on this strain is provided in the Supporting Information. The mammalian cells employed were Chinese hamster ovary (CHO) cell line AS52, clone 11-4-8.39−41 CHO cells were maintained in Ham’s F12 medium containing 5% FBS, 1% antibiotics (100 U/mL sodium penicillin G, 100 μg/mL streptomycin sulfate, 0.25 μg/mL amphotericin B in 0.85% saline), and 1% glutamine at 37 °C in a humidified atmosphere of 5% CO2. Mutagenicity in S. typhimurium. A single colony isolate of YG7108 was transferred into 100 mL of Luria−Bonner (LB) medium supplemented with kanamycin (25 μg/mL) and chloramphenicol (10 μg/mL). The culture was grown overnight at 37 °C with shaking (200 rpm). The cells were harvested by centrifugation, washed in 100 mM potassium phosphate buffer (PPB, pH 7.4), and the titer was adjusted to 6 × 109 cells/mL. Treatments were conducted in 96-well round-bottom microplates. Each treatment well contained 3 × 108 cells, μL amounts of the chemical ±35 μL of S9 mix for a total volume of 100 μL. The S9 activation mix was prepared according to previously published methods;20,42 in the treatment well, the final concentrations were 3.5 mM MgCl2, 10.5 mM KCl, 1.75 mM glucose-6-phosphate, 1.4 mM NADP, 17.5 mM sodium phosphate buffer, pH 7.4, and 7% (v/v) S9 fraction. The wells of the microplate were covered with sterile AlumnaSeal and the plate was incubated while shaking (200 rpm) for 1 h at 37 °C. After the treatment, the entire

MATERIALS AND METHODS Chemicals and Reagents. Eleven nitramines and nitrosamines were analyzed in this study (Supporting Information, Table S1). Sigma-Aldrich N-nitrosodiethanolamine (99.8%), N-nitrosomorpholine, N-nitromorpholine (rare chemical library), and 1-nitro-4-nitrosopiperazine; Toronto Research Chemicals N-nitromonoethanolamine, 1-nitrosopiperazine, and 1,4-dinitrosopiperazine; Accustandard EPA method 521 nitrosamine standard mix; MP Biomedicals 1,4-dinitropiperazine; Chem Service N-nitrosodimethylamine (99.5%) were used as received. N-nitrodiethanolamine (92.4%), N-nitrodimethylamine (95%), and 1-nitropiperazine (95%) were synthesized by Dr. Bruce McKague of CanSyn Chem. Corp. (Toronto, ON, Canada). Synthetic procedures and techniques used to evaluate the purity of all nitrosamine and nitramine reagents were described previously.13 General reagents were purchased from Fisher Scientific Co. (Itasca, IL) and Sigma Chemical Co. (St. Louis, MO). Mammalian cell culture medium and fetal bovine serum (FBS) were purchased from Hyclone Laboratories (Logan, UT) or from Fisher Scientific Co. (Itasca, IL). S9 fraction was purchased from Molecular Toxicology (Annapolis, MD) and was a suspension of hepatic postmitochondrial supernatant isolated from Aroclor 1254-induced male Sprague−Dawley rat liver. Bacterial and Mammalian Cells. Salmonella typhimurium strain YG7108 was originally obtained from Dr. T. Nohmi, National Institute of Health Sciences, Tokyo, Japan. YG7108 was constructed from strain TA1535 (hisG46, rfa, and uvrB); in addition, this strain has deletions of ogt and ada that encode 8206

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contents of the well were mixed and added to 2 mL of molten Vogel−Bonner (VB) top agar supplemented with 550 μM histidine and biotin. The top agar was poured onto selective VB minimal medium plates, incubated for 72 h at 37 °C, and revertant his+ colonies were scored. For the determination of survivorship, an aliquot (10 μL) from each reaction well was removed. Following serial dilutions in PPB, the suspension was added to molten LB top agar and plated onto LB medium plates. These plates were incubated for 24 h at 37 °C and the resulting colonies were counted. Further details on the assay are provided in the Supporting Information. CHO Cell Chronic Cytotoxicity Assay. This assay measures the reduction in cell density on flat-bottom 96-well microplates as a function of the concentration of the chemical over a period of approximately 72 h (>3 cell cycles).43,44 Microliters of the sample in dimethyl sulfoxide (DMSO) were diluted with F12 + FBS medium. This assay was calibrated; the detailed procedure was published43,44 and is presented in the Supporting Information. For each chemical, four replicate wells were analyzed. The experiments were repeated two times. A concentration−response curve was generated for each chemical. A regression analysis was conducted with each curve. For each chemical, the LC50 value was calculated from the regression analysis; this represents the concentration that induced a 50% reduction in cell density as compared to the concurrent negative controls. CHO Cell Single Cell Gel Electrophoresis (SCGE) Assay. Single cell gel electrophoresis (SCGE, or Comet) assay quantitatively measures genomic DNA damage in individual nuclei induced by a test agent.45−47 We employed a microplate methodology46 with an enhanced hepatic microsomal mix (S9B150) specifically designed for use with nitrosamines;20 this consisted of 2 mM MgCl2, 6 mM KCl, 1.5 mM glucose-6-phosphate, 1.2 mM NADP, 10 mM sodium phosphate buffer, pH 7.4, 2 mM CaCl2, and 3.36% (v/v) S9 fraction. The SCGE metric for genomic DNA damage induced by the chemical was the % Tail DNA value, which is the amount of DNA that migrated from the nucleus into the microgel.48 Within each concentration range with >70% cell viability, a concentration−response curve was generated and regression analysis was used to fit the curve. The concentration inducing a 50% Tail DNA value was calculated from each concentration−response curve. A detailed description of the SCGE protocol is presented in the Supporting Information. Safety. Manipulations of toxic chemicals were conducted in certified Stage-2 containment biological/chemical safety hoods. This project was registered with and met the requirements of the University of Illinois.

Table 2. Comparative Chronic Cytotoxicity of Nitrosamines and Nitramines in CHO Cells

compound

conc. range (mM)

CHO cell chronic cytotoxicity (LC50) (mM)a

N-nitrosodimethylamine N-nitrodimethylamine N-nitrosomorpholine N-nitromorpholine N-nitrosodiethanolamine N-nitrodiethanolamine 1-nitrosopiperazine 1-nitropiperazine 1,4-dinitrosopiperazine 1,4-dinitropiperazine

0.005−10 0.0012−10 0.0003−10 0.0003−10 0.00009−12.5 0.00009−12.5 0.00009−12.5 0.0003−10 0.00009−12.5 0.0003−10

negative negative 11.1* 84.6* negative 12.6* 9.51 8.89 12.8* 2.85

r2b

CHO chronic cytotoxicity index valuec

NA NA 0.96 0.85 NA 0.78 0.99 0.99 0.98 0.97

0 0 90.1 11.8 0 79.3 105.1 112.4 78.1 350.8

a

The calculated test agent concentration that induced a cell density that was 50% of the negative control (LC50 value). The LC50 values designated with an asterisk were calculated by extrapolating concentration−response curves beyond the test chemical concentrations employed in the experiments. bCoefficient of determination of the goodness of fit for the regression analyses used for the LC50 calculation. cThis value was calculated as (LC50)−1(103); a larger value represents a greater cytotoxic potency. NA = not applicable.

N-nitrosodimethylamine and the 1,4-dinitrosopiperazine sample contained 0.9% 1-nitrosopiperazine. Mutagenicity of Nitrosamines and Nitramines in S. typhimurium. Metabolic activation of nitrosamines and nitramines is primarily dependent on cytochrome P-450, which converts them into DNA alkylating agents.49 Oxidative transformation is an important route of metabolism of xenobiotics. These oxidative reactions are largely catalyzed by the hepatic monooxygenase system based on cytochrome P-450. The monooxygenase system is formed by the enzyme system of cytochrome P-450 and NADPH cytochrome P-450 reductase. The hemoprotein cytochrome P-450 functions as the terminal oxidase involved in the hydroxylation of xenobiotics such as nitrosamines. In previous studies, the nitramines N-nitrodibutylamine, N-nitrodiethylamine, and N-nitrodimethylamine were activated by liver microsomes.50,51 In the present study, each chemical was analyzed for mutagenicity in S. typhimurium strain YG7108 under suspension test conditions with and without microsomal metabolic activation. Figure 1 illustrates the concentration−response curves for cytotoxicity (Figure 1A,C,E,G,I,K) and mutagenicity (Figure 1B,D,F,H,J,L). Most of the nitrosamines and nitramines required metabolic activation to exhibit mutagenicity. For each agent, a quantitative comparative mutagenicity analysis was conducted and the results are presented in Table 1. Mutagenic potency measures mutagenic activity as the number of induced mutants per micromole (μmol) of chemical; this is a sensitive method to compare the mutagenicity of test chemicals.52 S. typhimurium mutagenic potency values were derived from the concentration−response curves (Figure 1). The descending rank order of S. typhimurium mutagenic potency with S9 activation was N-nitrosodimethylamine ≫ N-nitrosomorpholine > N-nitrodimethylamine >1,4-dinitrosopiperazine ≫ N-nitromorpholine >1,4-dinitropiperazine > N-nitromonoethanolamine > N-nitrosodiethanolamine (Table 1). The range in mutagenic response was over 3 orders of magnitude. Weak direct-acting mutagenic responses (without S9 activation) were observed with N-nitrosodiethanolamine, N-nitrodiethanolamine, 1,4-dinitropiperazine, and N-nitromonoethanolamine. 1-Nitrosopiperazine

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RESULTS AND DISCUSSION Purity of Nitrosamines and Nitramine Analogues. Toxicity analyses may be impacted by cross-contamination by nitrosamine or nitramine analogues. For example, if N-nitrodimethylamine were 10 times less toxic than N-nitrosodimethylamine, but contained 10% N-nitrosodimethylamine as a cross-contaminant, the observed toxicity of the N-nitrodimethylamine may relate to the N-nitrosodimethylamine cross-contaminant. Gas or liquid chromatography mass spectrometry or HPLC-UV analyses were used to evaluate cross-contamination of all nitrosamine and nitramine analogues, as described previously.13 Overall, cross-contamination was N-nitrosomorpholine >1,4-dinitrosopiperazine (Table 3). These three agents were also the most potent nitrosamines in the S. typhimurium mutagenicity assay. Note that, in all cases, the nitramine was less mutagenic than its nitrosamine analogue. This was reflected by the CHO cell genotoxicity assay in that none of the nitramines induced a genotoxic response with or without metabolic activation. 1-Nitrosopiperazine and 1-nitropiperazine were negative with S. typhimurium as well as in the CHO SCGE assay. N-Nitrosodimethylamine (NDMA) Equivalency Relationship among the Nitrosamines and Nitramines. One approach to compare the relative level of the genotoxicity

N-nitrosamines exhibiting a mutagenic response (i.e., excluding 1-nitrosopiperazine) were promutagens; they either required cytochrome P450-mediated monooxygenase metabolic activation to be converted into their ultimate genotoxic forms, or, in the case of N-nitrosodiethanolamine, exposure to metabolic activation increased the mutagenic potency. Among N-nitramines exhibiting a mutagenic response (i.e., excluding 1-nitropiperazine), N-nitrodimethylamine and N-nitromorpholine were promutagens, requiring cytochrome P450-mediated monooxygenase metabolic activation to exhibit mutagenicity. However, 1,4-dinitropiperazine, N-nitromonoethanolamine, and N-nitrodiethanolamine did not require metabolic activation. Among structures exhibiting a mutagenic response, the N-nitrosamine analogue was generally significantly more mutagenic than the N-nitramine analogue (i.e., 13-fold for N-nitroso/nitrodimethylamine, 25-fold for N-nitroso/ nitromorpholine, and 7-fold for 1,4-dinitroso/nitropiperazine). Chronic Cytotoxicity of Nitrosamines and Nitramines in CHO cells. Direct chronic CHO cell cytotoxicity was evaluated and, in general, these agents were not highly cytotoxic (Table 2). The LC50 values were determined from the concentration−response curves for each agent. Reflecting their low cytotoxic potencies, for some chemicals, a 50% reduction in cell density compared to the negative control was not observed over the concentration range evaluated; for these chemicals, the LC50 value was estimated by extrapolation of the data. As a group, the piperazines were more cytotoxic. Although the toxicological mechanism for this direct-acting activity is not understood, it is interesting to note that the nitramines associated with diethanolamine and piperazine were more toxic than their nitrosamine analogues, and that the most toxic agents are ring-containing chemical structures. 8209

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Systems (Water CAMPWS) a National Science Foundation Science and Technology Center, under Award CTS-0120978.

among this group of chemicals is to calculate the mutagenic potency of nitrosamines and nitramines as a function of the mutagenicity of NDMA. NDMA is a regulated environmental agent that has an established human health risk standard. The U.S. EPA IRIS database indicates that a drinking water concentration of 0.7 ng/L of N-nitrosodimethylamine is associated with a 1 in 1 million excess lifetime cancer risk.25 The nitrosamines are genotoxic carcinogens, and several national and regional governmental agencies have set limits for NDMA.25,26,53 The S. typhimurium mutagenic potencies (on a per mole basis) were converted into NDMA equivalents (Figure S9 in the Supporting Information and Table 4). Data are included for two additional

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(1) IPCC Summary for Policymakers. In Climate Change 2013: The Physical Science Basis; Cambridge University Press: Cambridge, U. K., 2013; p 28. (2) International Energy Agency. Topics: Carbon capture and storage, 2013. (3) International Energy Agency. Tracking Progress in Carbon Capture and Storage: International Energy Agency/Global CCS Institute report to the third Clean Energy Ministerial; OECD/International Energy Agency and Global CCS Institute: Paris, 2012; p 20. (4) Statoil. http://www.statoil.com/en/TechnologyInnovation/ NewEnergy/Co2Management/Pages/CarbonCapture.aspx. (5) Fostas, B.; Gangstad, A.; Nienseter, B.; Pedersen, S.; Sjovoll, M.; Sorensen, A. L. Effects of NOx in the flue gas degradation of MEA. Energy Procedia 2011, 4, 1566−1573. (6) Lackner, K. S. Capture of carbon dioxide from ambient air. Eur. Phys. J.: Spec. Top. 2009, 176, 93−106. (7) Reynolds, A. J.; Verheyen, T. V.; Adeloju, S. B.; Meuleman, E.; Feron, P. Towards commercial scale postcombustion capture of CO2 with monoethanolamine solvent: key considerations for solvent management and environmental impacts. Environ. Sci. Technol. 2012, 46 (7), 3643−3654. (8) Challis, B. C.; Kyrtopoulos, S. A. Chemistry of nitrosocompounds. Part 11. Nitrosation of amines by the 2-phase interaction of amines in solution with gaseous oxides of nitrogen. J. Chem. Soc., Perkin Trans. 1 (1972-1999) 1979, 1 (2), 299−304. (9) Dai, N.; Mitch, W. A. Effects of flue gas compositions on nitrosamine and nitramine formation in post-combustion CO2 capture systems. Environ. Sci. Technol. 2014, No. 10.1021/es501864a. (10) Goldman, M. J.; Fine, N. A.; Rochelle, G. T. Kinetics of Nnitrosopiperazine formation from nitrite and piperazine in CO2 capture. Environ. Sci. Technol. 2013, 47 (7), 3528−3534. (11) Dai, N.; Mitch, W. A. Influence of amine structural characteristics on N-nitrosamine formation potential relevant to postcombustion CO2 capture systems. Environ. Sci. Technol. 2013, 47 (22), 13175−13183. (12) Shah, A. D.; Dai, N.; Mitch, W. A. Application of ultraviolet, ozone, and advanced oxidation treatments to washwaters to destroy nitrosamines, nitramines, amines, and aldehydes formed during aminebased carbon capture. Environ. Sci. Technol. 2013, 47 (6), 2799−2808. (13) Dai, N.; Shah, A. D.; Hu, L.; Plewa, M. J.; McKague, B.; Mitch, W. A. Measurement of nitrosamine and nitramine formation from NOx reactions with amines during amine-based carbon dioxide capture for postcombustion carbon sequestration. Environ. Sci. Technol. 2012, 46 (17), 9793−9801. (14) Hanst, P. L.; Spence, J. W.; Miller, M. Atmospheric chemistry of N-nitroso dimethylamine. Environ. Sci. Technol. 1977, 11 (4), 403− 405. (15) Pitts, J. N., Jr; Grosjean, D.; Van Cauwenberghe, K.; Schmid, J. P.; Fitz, D. R. Photooxidation of aliphatic amines under simulated atmospheric conditions: formation of nitrosamines, nitramines, amides, and photochemical oxidant. Environ. Sci. Technol. 1978, 12 (8), 946−953. (16) Plumlee, M. H.; Reinhard, M. Photochemical attenuation of Nnitrosodimethylamine (NDMA) and other nitrosamines in surface water. Environ. Sci. Technol. 2007, 41 (17), 6170−6176. (17) Hutchings, J. W.; Ervens, B.; Straub, D.; Herckes, P. Nnitrosodimethylamine occurrence, formation and cycling in clouds and fogs. Environ. Sci. Technol. 2010, 44 (21), 8128−8133. (18) Shah, K. A.; Karnes, H. T. A review of the analysis of tobaccospecific nitrosamines in biological matrices. Crit. Rev. Toxicol. 2010, 40 (4), 305−327. (19) Jakszyn, P.; Agudo, A.; Ibanez, R.; Garcia-Closas, R.; Pera, G.; Amiano, P.; Gonzalez, C. A. Development of a food database of nitrosamines, heterocyclic amines, and polycyclic aromatic hydrocarbons. J. Nutr. 2004, 134 (8), 2011−2014.

Table 4. NDMA Equivalent Values of the Nitrosamines and Nitramines Derived from the S. typhimurium Mutagenic Potency Values nitrosamine or nitramine

NDMA equiv value

N-nitrosodimethylamine N-nitrodimethylamine N-nitrosomorpholine N-nitromorpholine N-nitrosodiethanolamine 1-nitrosopiperazine 1-nitropiperazine 1,4-dinitrosopiperazine 1,4-dinitropiperazine N-nitrosopyrrolidine N-nitrosopiperidine N-nitromonoethanolamine

1 0.077 0.311 0.013 0.001 0 0 0.058 0.009 0.046 0.448 0.006

nitrosamines, N-nitrosopyrrolidine and N-nitrosopiperidine, which are drinking water disinfection byproducts. These results demonstrate a wide range of responses among the nitrosamines and nitramines and that NDMA is the most mutagenic. Clearly, additional research is needed in order to generate accurate human health risk estimates. The data generated in this study suggests that byproducts of amine-based carbon sequestration should not be regulated as a class but as individual compounds.

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ASSOCIATED CONTENT

S Supporting Information *

Description of the nitrosamine and nitramine agents analyzed, in vitro biological analytical methods, interpretation, and a discussion on the NDMA equivalent methodology. This material is available free of charge via the Internet at http://pubs.acs.org.

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REFERENCES

AUTHOR INFORMATION

Corresponding Author

*M. J. Plewa. Phone: (217) 333-3614. E-mail: mplewa@illinois. edu. Address: University of Illinois, 364 NSRC, 1101 West Peabody Drive, Urbana, IL 61801, United States. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work, funded by the Norwegian State, has been part of an extensive investigation program with the objective of developing methods and procedures for evaluating health and environmental impact of amine-based solvents under the CO2 Capture Mongstad project (CCM), organized as a joint effort by Gassnova SF and Statoil ASA. We appreciate support by the Center of Advanced Materials for the Purification of Water with 8210

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