Environ. Sci. Technol. 2010, 44, 3369–3375
Application of an Optimized Total N-Nitrosamine (TONO) Assay to Pools: Placing N-Nitrosodimethylamine (NDMA) Determinations into Perspective PANKAJ KULSHRESTHA,† KATHERINE C. MCKINSTRY,† BERNADETTE O. FERNANDEZ,‡ MARTIN FEELISCH,‡ AND W I L L I A M A . M I T C H * ,† Department of Chemical Engineering, Environmental Engineering Program, Yale University, New Haven, Connecticut 06520, and Warwick Medical School, University of Warwick, Coventry, CV4 7AL, United Kingdom
Received February 1, 2010. Revised manuscript received March 13, 2010. Accepted March 19, 2010.
Although N-nitrosodimethylamine (NDMA) has been the most prevalent N-nitrosamine detected in disinfected waters, it remains unclear whether NDMA is indeed the most significant N-nitrosamine or just one representative of a larger pool of N-nitrosamines. A widely used assay applied to quantify nitrite, S-nitrosothiols, and N-nitrosamines in biological samples involves their reduction to nitric oxide by acidic tri-iodide, followed by chemiluminescent detection of the evolved nitric oxide in the gas phase. We here describe an adaptation of this method for analyzing total N-nitrosamine (TONO) concentrations in disinfected pools. Optimal sensitivity for N-nitrosamines was obtained using a reduction solution containing 13.5 mL glacial acetic acid and 1 mL of an aqueous 540 g/L iodide and 114 g/L iodine solution held at 80 °C. The method detection limit for N-nitrosamines was 110 nM using 100 µL sample injections and NDMA as a standard. N-nitrosamines featuring a range of polarities were converted to nitric oxide with 75-103% efficiency compared to NDMA. Evaluation of potential interfering species indicated that only nitrite and S-nitrosothiols were a concern, but both interferences were effectively eliminated using group-specific sample pretreatments previously employed for biological samples. To evaluate the low TONO concentrations anticipated for pools, 1 L samples were extracted by continuous liquid-liquid extraction with ethyl acetate for 24 h, and concentrated to 1 mL. N-nitrosamine recovery during extraction ranged from 37-75%, and there was a potential for artifactual nitrosation of amines during solvent reflux in the presence of significant nitrite concentrations, but not at the low nitrite concentrations prevalent in most pools. Using the 1000fold concentration factor and 56% average extraction efficiency, the method detection limit would be 62 pM (5 ng/L as NDMA). The TONO assay was applied to six pools and their common tap water source in conjunction with analysis for specific * Corresponding author phone: (203) 432-4386; fax: (203) 4324387; e-mail:
[email protected]. † Yale University. ‡ University of Warwick. 10.1021/es100361f
2010 American Chemical Society
Published on Web 03/31/2010
nitrosamines. Even accounting for the range of N-nitrosamine extraction recoveries, NDMA accounted for an average of only 13% (range 3-46%) of the total nitrosamine pool.
Introduction The formation of N-nitrosodimethylamine (NDMA) as a disinfection byproduct has caused considerable concern since its discovery in the 1990s (1). Interest in N-nitrosamine formation is likely to grow, because nitrosamines are potent carcinogens, and because two trends in the drinking water industry are likely to promote their formation. First, utilities are increasingly exploiting algal- or wastewater-impacted waters to meet growing water demands. Such waters feature elevated concentrations of organic nitrogen (2), which can serve as nitrosamine precursors. Indeed, NDMA formation is particularly associated with wastewater-impacted waters (3, 4). Second, as a result of the more stringent effluent limitations on trihalomethanes (THMs) and haloacetic acids (HAAs) embodied in the Disinfection By-Product Rules of the U.S. Environmental Protection Agency (U.S. EPA) (http:// www.epa.gov/safewater/disinfection/stage2/index.html), drinking water utilities are incorporating chloramination for secondary disinfection. Reaction pathway studies have indicated that nitrosamine formation can arise from a reaction between amine precursors with dichloramine, which always co-occurs with monochloramine during chloramination (5). The U.S. EPA has classified several nitrosamines as probable human carcinogens and placed them on the Contaminant Candidate List 3 (www.epa.gov/safewater/ccl). The detection of other N-nitrosamines in disinfected waters has been pursued. N-nitrosodiethylamine, N-nitrosopyrrolidine, and N-nitrosomorpholine were detected in disinfected wastewater effluents (4, 6). N-nitrosodiethylamine, N-nitrosopiperidine, N-nitrosodiphenylamine, Nnitrosomorpholine, and N-nitrosopyrrolidine, were detected in chloraminated drinking waters in Canada (7-9). Nnitrosodibutylamine was detected in disinfected pools (10). In nearly all cases, NDMA concentrations have far exceeded those of other N-nitrosamines, suggesting that NDMA may dominate the nitrosamine pool. However, the list of targeted N-nitrosamines has largely been limited to commercially available, low molecular weight nitrosamines on the U.S. EPA’s Integrated Risk Information System (11), regardless of whether precursors for these particular N-nitrosamines are likely to occur in source waters or not. There are several reasons to expect that N-nitrosamines other than NDMA may be important. While reaction pathway studies have focused on NDMA formation from dimethylamine, they indicate that conversion of secondary amines to their respective N-nitrosamines (e.g., morpholine to N-nitrosomorpholine) is anticipated (5). Furthermore, tertiary amines rapidly degrade to secondary amines during chlorination or chloramination (12), while quaternary amines undergo similar conversions at much lower yields (13). Hence, the array of potential N-nitrosamines is limited only by the variety and abundance of secondary, tertiary, and quaternary amine precursors in waters. Concentrations of dimethylamine and trimethylamine precursors for NDMA are generally 600 byproducts, 70% of TOX remains unidentified. A total N-nitrosamine (TONO) assay would indicate whether NDMA and other specific nitrosamines of current interest are dominant or minor components of the total N-nitrosamine pool. The TONO assay described here is based on a nonchromatographic method developed previously to quantify nitrite and the total concentrations of several categories of nitrosated species, including N-nitrosamines, S-nitrosothiols, and nitrosohemes in blood and other biological matrices (17-19). S-nitrosothiols (RSNO) serve as temporary stores of nitric oxide, a signaling molecule that also binds to hemoglobin and other ferrous heme complexes, and both S-nitrosothiols and nitrosohemoglobin are believed to fulfill important cell signaling functions in biology. In contrast, the biological significance of N-nitroso species in blood and tissues (20) is unclear. In this paper, we optimize this assay for application to disinfected waters. Combined with analysis for specific N-nitrosamines, we apply the TONO assay to evaluate the relative importance of NDMA to the total N-nitrosamine pool within a range of disinfected pools, as well as the tap water serving as their common source water. Pools are of interest for several reasons. First, an epidemiological study has found that, like drinking chlorinated water, swimming in chlorinated pools was associated with an increased risk of bladder cancer (21); further studies are needed to confirm these findings. Second, these waters represent extreme cases of disinfection, featuring elevated disinfectant concentrations (e.g., 1-3 mg/L chlorine as Cl2) over average water residence times of several months. In addition to humic substances in the source water, bathers contribute precursors from urine, sweat, saliva, hair, and personal care products such as cosmetics (22). These latter classes of precursors are often nitrogen-rich. Lastly, we have evaluated the concentrations of NDMA and other low molecular weight N-nitrosamines in a series of pools (10). NDMA concentrations increased from ∼6 ng/L in outdoor pools, where sunlight photolysis was possible, to ∼30 ng/L in indoor pools and ∼300 ng/L in indoor hot tubs. NDMA concentrations exceeded those of other N-nitrosamines by at least an order of magnitude. The risk posed by dermal exposure to nitrosamines remains an open question.
Materials and Methods Optimized Method. Sources of materials are provided in the Supporting Information. Tap and recreational water samples were collected in fluorinated high-density polyethylene containers, and returned immediately to the laboratory for measurement of temperature and total residual chlorine by the N,N-diethyl-p-phenylenediamine (DPD) colorimetric method (23). An aliquot was reserved for total organic carbon (TOC) analysis using a Shimadzu TOC-VCSH total organic carbon analyzer. The disinfectant residual was quenched with 200 mg/L ascorbic acid. Aliquots were analyzed for nitrite 3370
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by a standard colorimetric method (23) and the samples were stored at 4 °C pending further analysis. Aliquots (500 mL) were analyzed in duplicate for specific N-nitrosamines by gas chromatography-tandem mass spectrometry in the chemical ionization mode with methanol (GC/CI/MS/MS) according to U.S. EPA Method 521 (http://www.epa.gov/ nerlcwww/ordmeth.htm). Aliquots (1 L) were extracted in duplicate by continuous liquid-liquid extraction over 24 h with 400 mL ethyl acetate using Aldrich liquid-liquid continuous extractors. The ethyl acetate extracts were concentrated to 5 mL by rotary evaporation, and blown down to 1 mL under nitrogen gas. To improve solubility in the reactor chamber, the ethyl acetate extract was supplemented with 1 mL isopropanol before concentration to 1 mL. A glass reactor was constructed consisting of a jacketed cylindrical reaction chamber (18 mL) surmounted by a condenser (Supporting Information Figure SI-1). A glass frit at the bottom of the chamber permitted the bubbling of ultra high purity nitrogen gas. A drain and a sample injection port capped by a septum were located at the chamber bottom. Via external jackets, the reactor vessel was maintained at 80 °C, while the condenser was maintained at -20 °C by a 50/ 50 mixture of water and antifreeze. While purging with nitrogen, the reactor chamber was filled with 13.5 mL glacial acetic acid, and 1 mL of an aqueous tri-iodide solution consisting of 540 g/L potassium iodide and 114 g/L iodine. The tri-iodide solution was made fresh daily, as older solutions tended to increase the baseline signal. The reaction solution was purged for several minutes prior to connecting the condenser. The acidic iodine/iodide mixture forms reducing agents, such as HI and HI3, that release nitric oxide (NO) from four types of species via the following net reactions (19): nitrite: 2 HI + 2 NO2 f I2 + 2 NO + 2 OH
N-nitrosamines (R2NNO): 2 HI + 2 R2NNO f I2 + 2 NO + 2 R2NH
(1)
(2)
S-nitrosothiols(RSNO): 2 HI + 2 RSNO f I2 + 2 NO + 2 RSH (3) nitrosohemoglobin (Hb-Fe(II)-NO): 2 HI + 2 Hb-Fe(II)-NO f I2 + 2 NO + 2 Hb-Fe(II)
(4)
To eliminate interfering signals from S-nitrosothiols and nitrite, the 1 mL sample concentrates were treated with 100 µL of a 20 g/L solution of mercuric chloride in deionized water in the dark for 30 min at room temperature. This treatment destroys S-nitrosothiols according to eq 5 (19, 24). Hg2+ + 2 RSNO + 2 H2O f Hg(RS)2 + 2 HNO2 + 2 H+ (5) The solution was then treated for 15 min in the dark with 100 µL of a 50 g/L solution of sulfanilamide in 1 N hydrochloric acid to destroy nitrite by converting it to a diazonium cation (19, 24). A treated sample aliquot (generally 100 µL) was then introduced into the reaction vessel. The nitric oxide released was purged from the vessel and condenser by the nitrogen gas stream, and passed through a 1 M sodium hydroxide trap held within an ice bath, a gas pressure meter, and a 0.22 µm syringe filter prior to entering an Ecomedics CLD 88 sp chemiluminescence detector. The gas pressure was maintained at 0-1 psi gauge to permit sample introduction without fracturing internal detector components. Within the detector, the NO is oxidized to nitrogen dioxide (NO2) by ozone. Part of this NO2 arises in an electronically excited state (NO2*). The light emitted while reverting to the ground state is quantified by a photomultiplier, and the chemiluminescence
TABLE 1. Reaction Condition Optimization R2
KI (g/L)
I2 (g/L)
108 324 216 324 540 756 324 540
38 76 114 114 114 114 190 190
temperature 60 °C 17 0.99 24 0.99 54 0.98 33 0.98 61 0.96 24 0.95 42 0.99 40 0.98
1-10 µM 1-10 µM 1-10 µM 1-10 µM 1-10 µM 1-10 µM 1-10 µM 1-10 µM
216 324 540
114 114 114
temperature 70 °C 33 0.99 25 0.98 71 0.96
1-10 µM 1-10 µM 1-10 µM
114 114 114
temperature 80 °C 42 0.99 28 0.99 164 0.99
0.1-1 µM 0.1-1 µM 0.1-1 µM
216 324 540
slope
TABLE 2. Model Compound Recovery replicate recovery (%)a
standard curve range
1
2
3
averageb
N-nitrosamines nitrosodimethylamine nitrosomorpholine nitrosodibutylamine nitrosodiphenylamine nitrosodiethanolamine U.S. EPA 521 mixc
100 97 99 88 74 100
100 94 102 89 75 104
100 98 101 92 76 105
100 (0) 96 (2) 101 (2) 90 (2) 75 (1) 103 (3)
C-nitroso compounds 4-nitrosophenol
