1. Disinfection Byproducts in Drinking Water and Human Health Dave Reckhow University of Massachusetts - Amherst 2009 GRC on Water Disinfection By-Products

2. Outline DBP Discovery – Complementary approaches What’s new? – Iodo compounds – N-DBPS Reactivity of Specific Nitrogenous Constituents – Amino Acids – Amines, Purines & Pyrimidines – Others What next? Initial products & End products Focus on reactions with free chlorine, including comments on other disinfectants

3. 3 John #1: Dr. John Snow Cholera – First emerged in early 1800s – 1852-1860: The third cholera pandemic Snow showed the role of water in disease transmission – London’s Broad Street pump (Broadwick St) Miasma theory was discredited, but it took decades to fully put it to rest 1813-1858 2007

4. 4 Picadilly Circus Soho, Westminster

5. John #2: Dr. John L. Leal 5 Jersey City’s Boonton Reservoir Leal experimented with chlorine, its effectiveness and production – George Johnson & George Fuller worked with Leal and designed the system (1908) “Full-scale and continuous implementation of disinfection for the first time in Jersey City, NJ ignited a disinfection revolution in the United States that reverberated around the world” M.J. McGuire, JAWWA 98(3)123 1858-1914

6. Leal on chlorine “the practical application of the use of bleach (chlorine) for the disinfection of water supplies seems to me to be a great advance in the science of water purification. It is so cheap, so easy and quick of application, so certain in its results, and so safe, that it seems to me to cover a broader field than does any other system of water purification yet used.” – John L. Leal, 1909 6

7. 7 Chlorination 1-2 punch of filtration & chlorination Melosi, 2000, The Sanitary City, John Hopkins Press Greenberg, 1980, Water Chlorination, Env. Impact & Health Eff., Vol 3, pg.3, Ann Arbor Sci. US Death Rates for Typhoid Fever

8. John #3: Johannes J. Rook Short Biography – Education PhD in Biochemistry: 1949 – Work experience Technological Univ., Delft (~‘49-’54) – Laboratory for Microbiology Lundbeck Pharmaceuticals in Copenhagen, (~’55-?) Noury Citric acid Factory (in Holland) Amstel Brewery Rotterdam Water Works by 1963, chief chemist (1964-1984). 1984-1986; Visiting Researcher at Lyonnaise des Eaux, Le Pecq. – Early Research 1955, Microbiological Deterioration of Vulcanized Rubber – Applied Micro. 1964, secured funds for a GC at Rotterdam – Carlo Erba with gas sample loop 8

9. 9 John Rook & DBPs Major Contributions – Brought headspace analysis from the beer industry to drinking water T&O problems – Found trihalomethanes (THMs) in finished water Carcinogens !?! – Published in Dutch journal H2O, Aug 19, 1972 issue – Deduced that they were formed as byproducts of chlorination – Proposed chemical pathways Rook, 1974, Water Treat. & Exam., 23:234

10. Reactions with Disinfectants: Chlorine 10 HOCl + natural organics (NOM) Oxidized NOM and inorganic chloride Aldehydes Chlorinated Organics TOX THMs HAAs The THMs The Precursors!

11. The Haloacetic Acids 11 HAA5 & HAA6 include the two monohaloacetic acids (MCAA & MBAA) plus – One of the trihaloacetic acids: – And 2 or 3 of the dihaloacetic acids HAA6 only 11

12. Haloacetonitriles 12 Others that are commonly measured, but not regulated include the: – Dihalo- acetonitriles – Trihaloacetonitriles 12

13. Halopropanones 13 As well as the: – dihalopropanones – trihalopropanones 13

14. 14 DBPs: Formation in Plant Dist. Sys. Cl 2 Coagulant Cl 2 NH 3 Settling Filtration Nitrosamines Trihalomethanes Haloacetic Acids Dave Reckhow, UMass-Amherst

15. 15 Epidemiology Bladder Cancer – DBPs linked to 9,300 US cases every year Other Cancers – Rectal, colon Reproductive & developmental effects – Neural tube defects – Miscarriages & Low birth weight – Cleft palate Other – Kidney & spleen disorders – Immune system problems, neurotoxic effects 137,000 at risk in US?

16. 16 National Distribution 241,000,000 people in US are served by PWSs that apply a disinfectant Gray et al., 2001 [Consider the Source, Environmental Working Group report] High THMs are levels of at least 80 ppb over a 3 month average

17. Hunting for the bad DBPs Observational/empirical – Multifaceted analysis of treated waters – Companion toxicity testing Deductive/theoretical – Postulate DBPs from known NOM substructures – Exploit Structure-toxicity models Also allows us to probe NOM contributions to regulated DBPs

18. The DBP Iceberg Halogenated Compounds Non-halogenated Compounds ICR Compounds 50 MWDSC DBPs ~700 Known DBPs THMs, THAAs DHAAs Stuart Krasner Susan Richardson

19. 19 Total Organic Halogen Standard Methods; USEPA Method #1650 Activated Carbon Adsorption & Pyrolysis & Microcoulometric Detection of halide Extended Method for TOCl, TOBr, TOI Trap gases & ion chromatography – (e.g., Hua & Reckhow, 2008) Pyrolysis Oven Microcoulometric Cell GAC Adsorption

20. The TOX Pie

21. TOX Distribution of Newport News Water Hua & Reckhow, 2007

22. MW Distribution of Unknown TOX Hua & Reckhow, 2007 Substantial overestimation of MW due to charge effects

23. Chlorine & Ozone produce iodate Cambridge MA Water, DOC: 4.2 mg/L, I: 200  g/L Hua & Reckhow, 2007

24. Iodinated TOX (TOI) Cambridge MA Water, DOC: 4.2 mg/L, I: 200  g/L TOI : NH 2 Cl > ClO 2 > Cl 2 > O 3 Hua & Reckhow, 2007

25. Regulated DBP as surrogates EPA’s ICR Database

26. Organic Chloramines Stable N-chloroaldimine from amino acids – Pathway favored at lower pHs Half-life of 35-60 hrs @pH 7-8 Conyers & Scully, 1993 [ES&T 27:261] Boning Liu PhD student

27. TOX pie revised Organic Chloramines Median C/N ratio (15)

28. 28 Source: Who is really responsible? or

29. 29 Watershed Origins Aquifer Lake Sediment & Gravel in Lake Bed Algae

30. 30 Leaching Experiments White Pine Red Maple White Oak Darleen Bryan’s study

31. 31 Algae as THM Precursors From: Plummer & Edzwald, 2001 – [ES&T:35:3661] Scenedesmus quadricauda Cyclotella sp. ~25% from EOM pH 7, 20-24ºC, chlorine excess Algae

32. Regulated DBP as surrogates EPA’s ICR Database

33. Watershed Origins 33 Aquifer Lake Upper Soil Horizon Lower Soil Horizon Sediment & Gravel in Lake Bed Litter Layer Algae 33

34. Plant biopolymers Cellulose Lignin – Phenyl-propane units – Cross-linked – Radical polymerization – Ill defined structure Hemicellulose Terpeniods Proteins

35. Lignin Monomers Aromatic structures from CuO degradation – Syringyl – Vanillyl – Cinnamyl

36. 4-hydroxy benzenes Among the most reactive structures tested

37. Alkali CuO oxidation Method Oven method: Hedges and Ertel (1982) – 1g CuO, 25-100 mg FAS, 7 mL NaOH – 170 o C in oven for 3 hours Microwave method: Goni (1998) – 500 mg CuO, 50 mg FAS, 15 ml 2N NaOH – 150 o C in microwave for 90 min

38. Mining the literature to postulate “new” DBPs Chlorination of p- hydroxybenzoic acid based on Larson and Rockwell (1979). “A” represents electrophilic aromatic substitution, “B” is oxidative decarboxylation Haloquinones are likely intermediates

39. PAHA II Fill in missing steps by analogy – Halohydroxy- dienoic acids – TCAA

40. Nitrogenous Biopolymers Why focus on these? – Nitrogenous organics are generally quite reactive – N-DBP formation can be enhanced by chloramination – Some evidence that they are major contributors to adverse human health effects of DBPs – Relatively little is known about N-DBPs Key suspects – Amino Acids & Proteins – Nucleic Acids, Pyrimidines & Purines – Others (e.g., porphyrins)

41. Organic Nitrogen Abundance Ratio to carbon – Redrawn from Westerhoff & Mash, 2002

42. N-DBPs we know about: end products Certain to come from N-organics when using free chlorine Major types: – Cyanogen Halides – Haloacetonitriles – Halonitromethanes Special focus on these compounds because of large data set CNCl & CNBr 9 species

43. Occurrence DHANs are typically 10% of THM level Krasner et al., 2002 [WQTC] – 12 plant survey ICR (mean for all) – HAN4: 2.7 µg/L – CP: <0.5 µg/L – CNCl: 2.1 µg/L

44. Work of Michael Plewa 44 Genotoxicity

45. Lowest Observed Adverse Effect Level – AWWARF report by Bull et al., 2007 Quantitative Structure-Toxicity Models

46. DHAN Chemical Degradation in Distribution Systems Accelerated by chlorine and base

47. Hydrolysis and oxidation Proposed Rate Law for DCAN k 1 = 1.78 x10 -7 ±0.35 x10 -7 (s -1 ) k 2 = 3.42 ±0.31 (M -1 s -1 ) k 3 = 1.30 x 10 -1 ±0.08 x 10 -1 (M -1 s -1 )

48. DCAN half-life based on pH & HOCl At 20 C From Reckhow, Platt, MacNeill & McClellan, 2001 – Aqua 50:1:1-13 Degradation in DS observed to increase with increasing pH – ICR data: Obolensky & Frey, 2002

49. DCAD Formed from degradation of DCAN Readily halogenated – Only exists as N-Cl-DCAD?

50. DCAD Stability

51. Organic Nitrogen Abundance Ratio to carbon – Redrawn from Westerhoff & Mash, 2002

52. Organic-N: Types & Abundance Amino Abun- Acid dance Glycine 11.7% Alanine 11.7% Valine 5.2% Isoleucine 4.4% Leucine 5.6% Serine 10.8% Threonine 7.0% Methionine 1.0% Aspartic Acid 10.2% Glutamic Acid 10.0% Lysine 1.6% Ornithine 2.0% Arginine 7.4% Histidine 2.5% Asparagine 0.4% Glutamine 0.5% Tryptophan 2.5% Phenylalanine 3.4% Tryrosine 2.2% SUM100.0% 52 [1] [1] Based on an average of: Isle Bay water (Yamashita & Tanoue, 2003), Lake Biwa (Wu et al., 2003), and water column values, summer, from Thomas, 1997 Classification50%ile90%ile99%ile DON3508002000 Free AA2050200 Combined AA40100400 Nucleic acids2050200 Amino Sugars40100400 Humic-N252001000 Estimates in µg-N/L From: Bull et al., 2006

53. Reaction pathways General scheme for carbonyl and cyano formation from chlorination of amines, amino acids & related compounds – (adapted from Nweke and Scully, 1989, and Armesto et al., 1998). Many N-halo products

54. 54 Total Organic Halogen Standard Methods; USEPA Method #1650 Activated Carbon Adsorption & Pyrolysis & Microcoulometric Detection of halide Extended Method for TOCl, TOBr, TOI Trap gases & ion chromatography – (e.g., Hua & Reckhow, 2008) Pyrolysis Oven Microcoulometric Cell GAC Adsorption

55. TOX pie revised N-Halo Organics Median C/N ratio (15)

56. Amino Acids Free, combined & “humic”

57. Amino Acids: Chlorine Demand More reactive than most NOM From: “DBP Formation from Amino Acids and Proteins in NOM”, Kim & Reckhow, in preparation

58. Classic Mechanism 2 Pathways – Formation of nitriles with excess chlorine – Formation of aldehydes with under-chlorination Aldehyde Froese, Kenneth L., Wolanski, Alina, and Hrudey, Steve E. “Factors Governing Odorous Aldehyde Formation as Disinfection By- Products in Drinking Water”. Water Research 33[6], 1355-1364. 1999. Isoleucine Nitrile

59. Dihaloacetic Acid Aspartic acid Histidine Asparagine

60. Dihaloacetonitriles Aspartic acid Histidine

61. Early Recognition of DHAN pathway – From Trehy, 1980 (MS Thesis) Aspartic Acid

62. Phenylalanine Stable N-chloroaldimine – Pathway favored at lower pHs Half-life of 35-60 hrs @pH 7-8 Conyers & Scully, 1993 [ES&T 27:261]

63. Asparagine Oxidant Residuals and CNCl – 0.036 mM Asparagine, pH 7, 30 min – Shang, Gong & Blatchley, 2000 (Shang et al., 2000) Free Tri Di Mono Residual Chlorine (mg/L) Cyanogen Chloride (ug/L)

64. Asparagine Proposed degradation pathway – Showing major, non-trivial “stable” products

65. Polypeptide linkages Amide nitrogen thought to react slowly – About 5% under conditions used Based on Jensen et al., 1999 More reactive than expected – Demands calculated from component AAs both with and without amide reactivity 4 281573619265 # of AAs

66. Proteins vs Amino Acids Cont Comparison to constituent free AAs – same dihalo; more trihalo – Somewhat more TOX & UTOX

67. Aromatic Amines When R is electron withdrawing – Otherwise ring halogenation precedes N- chlorination – With some R both can occur – May also occur with R in ortho position Quite stable From study of Sufamethoxazole (Dodd & Huang, 2004)

68. Some New DBPs Compound50%ile90%ile99%ile N-Chloroiminoacetonitrile0.30.62.5 N-N-Dichloroaminoacetonitrile0.61.45.5 N-Chlorophenylacetaldimine5.51353 Concentrations in µg/L Finished Drinking Water Concentrations Assumptions – N-Chloroiminoacetonitrile and N-N-Dichloroaminoacetonitrile were derived only from Asparagine at yields of 40% and 60% respectively – N-Chlorophenylacetaldimine was only from phenylalanine at a yield of 50% – Proteins yielded 50% of component AA byproducts

69. Summary A broad range of nitrogenous organic compounds in natural waters are reactive with chlorine and produce both regulated and non-regulated DBPs – Amino acids are generally reactive – Proteins may be more important than previously thought – Nucleic acids (bases) are all quite reactive N-chloro DBPs may be important – Reactive; could be quite prevalent; maybe toxic? – Causes “hidden TOX” Chloramination may not be effective at reducing these byproducts Other DBPs that may be of concern include – Haloquinones – Halonitriles – Nitrosamines – Iodo compounds

70. Acknowledgements Richard Bull UMass Researchers – Guanghui Hua & Boning Liu – Junsung Kim & Hans Mentzen – Andrew MacNeill Sponsors – AWWA Research Foundation (now WRF) Project #2867; Report #91135 – Others: NSF, EPA

71. The End

72. TD 50 ’s of carcinogenic nitroso compounds vs. NOAEL Halo DBPs

73. Research needs GroupExampleOccurrenceToxicology Haloquinones2,6-dichloro-3-methyl-1,2- benzoquinone (DMBQ) 12 Organic N- haloamines Prioritize on range of stabilities and mutagenic activity 21 Alkaloidal nitrosamines N-nitrosonornicotine 1-N- oxide 12 Cyclopentenoic acids & MX-related 3,5-dichloro—1-hydroxy-4- ketocyclopent-2-enoic acid CMCF 1212 2121 Halonitriles2,3-Dichloropropenal (Carc) 2,3-dibromopropionitrile (DT) 11 Conclusions: from QSTR

74. 74 Known vs. Unknown Cl 2 BPs Reaction Time

75. 75 Known vs. Unknown Cl 2 BPs Dose effects

76. 76 Known vs. Unknown Cl 2 BPs pH effects

77. Proteins & Polypeptides Ratio of observed to predicted Amide linkages cause: – small decrease in chlorine demand – Mixed affect on TOX – Higher trihalo DBPs (THM, TCAA) – No DCAN Cl2 DemandTOXTHMTCAADCAA Unkn TOX Albumin0.731.062.832.170.661.02 Hemoglobin0.910.904.493.200.610.52 Lysozyme0.821.702.403.510.991.73 Insulin0.851.661.401.631.051.98

78. Conclusions: AAs & Proteins Aspartic acid alone may be responsible for a substantial amount of the DHANs in treated drinking waters Proteins are surprisingly reactive despite “recalcitrant” amide nitrogens Side chains are probably the site of most attack Tendency to form more trihalo DBPs as compared to free amino acids, but almost no HANs Similar amount of unknown TOX is formed despite relatively unreactive amide nitrogen Need to identify more “UTOX” N-chloro compounds should be investigated

79. Glycine Na & Olson, 2006 Proposed Pathways of N,N- Dichloroglycine Decay

80. Chlorination of Tryptophan – From Trehy, 1980 (MS Thesis) Similar mechanism for Tyrosine – Trehy et al., 1986 ES&T 20:1117 Tryptophan M/MPredictionObservation Cl 2 Dem415 DHAN10.01-0.02 TOX21.9 THM00.16 THAA00.11 DHAA00.06 UTOX00.85

81. Continued Reaction From Model compound studies

82. Conclusions: Amino Acids All 20 essential amino acids are highly reactive with chlorine Several react to form substantial amounts of “unknown” organic halide byproducts – Histidine, Asparagine, Tryptophan, Tyrosine, Aspartic Acid Only Tryptophan and tyrosine produce elevated levels of THMs and THAAs Aspartic acid produces very large yields of DHANs and DHAAs Aspartic acid alone is responsible for a substantial amount of the DHANs in treated drinking waters

83. Conclusions: Proteins Proteins are surprisingly reactive despite “recalcitrant” amide nitrogens Side chains are probably the site of most attack HANs are not produced by the bound Asp Tendency to form more trihalo DBPs as compared to free amino acids Similar amount of unknown TOX is formed despite relatively unreactive amide nitrogen Need to identify more “UTOX”

84. Acknowledgements Research Support – American Waterworks Association Research Foundation – National Science Foundation Many UMass students, post-docs, and collaborators – Richard Bull – Junsung Kim, Darlene Bryan, Gladys Makdissy, Cynthia Castellon Many Drinking Water Utilities

85. Compare with Model Compounds 85 Wide range in reactivity

86. Balance of formation & degradation Above pH 7.5 – DCAD is formed faster than it decomposes Below pH 7.0 – DCAD decomposition is faster – It is just a transient intermediate; present at some pseudo- steady state concentration

87. NOM Origins: Metabolic Pathways Pyruvate Acetate Water Soluble Acids Porphyrins Amino Acids Nucleic Acids Misc. N & S compounds Proteins Shikimic Acid Carbohydrates Saponifiable Liquids Unsaponifiable Liquids Mevalonic acid Terpenoids Steroids Flavonoids Aromatic Compounds From: Robinson, 1991 Activated non-N precursors Nitrogenousprecursors

88. Aromatic Amines Proposed degradation pathway for 3- amino benzoic acid.

89. Ranges of Org-N by types Estimates from literature surveys Classification50%ile90%ile99%ile DON3508002000 Free AA2050200 Combined AA40100400 Nucleic acids2050200 Amino Sugars40100400 Humic-N252001000 Others Order of magnitude estimates for organic nitrogen in surface waters (all values in µg-N/L)

90. Algae are known Precursors From: Plummer & Edzwald, 2001 – [ES&T:35:3661] Scenedesmus quadricauda Cyclotella sp. ~25% from EOM pH 7, 20-24ºC, chlorine excess Algae

91. Algal Impacts on Water Supplies 91 High photosynthetic activity 91 Many examples – e.g., Lake Lanier Source for Gwinnett Co.’s (GA) Lanier WTP Often attributed to release of proteinaceous material

92. Minor except for two – Tryptophan – Tyrosine AAs: THM formation

93. Cyanogen Halides Cyanogen Chloride Cyanogen Bromide All at 20 o C Pedersen & Mariñas, 2001 Xie & Reckhow, 1993 Roughly in agreement with Na & Olson

94. Nitrosamines NDMA: typically formed at greater levels with chloramination than with chlorination – Continues to form across DS? other nitrosamines (beyond NDMA) have been reported in chloraminated water Levels and mechanisms – Earlier work: Valentine & Weinberg – New mechanism: Mitch

95. One possible pathway to NDMA Role of Dichloramine and oxygen From: Walse & Mitch, 2008 [ES&T, 42:4:1032]

96. 96 Unnatural Precursors? Ranitidine (Zantac) – 63% conversion to NDMA Schmidt et al., 2006 [WQTC] – Introduced in 1981, largest selling prescription drug by 1988 Stomach ulcers and esophageal reflux – Mean concentration of 3000 ng/L estimated for raw municipal WW (national average) Sedlak 2005 AWWARF report – 450 ng/L formation in raw WW expected – Unknowns: how much does this persist in treatment and in the environment?

97. Abbreviations – DMD: dimethyldiazene – TMT: tetramethyltetrazene – FMMH: formaldehyde monomethylhydrazone – FDMH: formaldehyde dimethylhydrazone – DMC: dimethylcyanamide – DMF: dimethylformamide From: Mitch & Sedlak, 2002 [ES&T, 36:588] Many products from UDMH

98. Reactions with Chlorine HOCl + natural organics (NOM) Oxidized NOM and inorganic chloride Aldehydes Chlorinated Organics TOX THMs HAAs The THMs The Precursors!

99. An Aquatic Humic “Structure” From Thurman, 1985

100. TOX: Known & Unknown Data from the Mills Plant (CA) August 1997 (courtesy of Stuart Krasner) Regulated DBPs But, the Bad Stuff is probably somewhere here

101. N-chloro-organics Reactions of chlorine with organic amines – Primary amines – Secondary amines Inorganic chloramines can transfer their active chlorine in a similar fashion

102. Degradation of Organic Chloramines

103. 103 Known vs. Unknown Cl 2 BPs Dose effects

104. Vanillins Similar patterns

105. Syringyls No ring-based THMs

106. Analysis of Lignins & NOM Multiple tests – Yields (mg/100mg)

107. DBP yields Lignin acts much like the sum of its monomers Substantial source of carbonaceous precursors From: “Analysis of Lignin in NOM Using Alkali CuO Oxidation”, Kim & Reckhow, in preparation

108. Why N-DBPs? Nitrogenous organics are generally quite reactive Can be enhanced by chloramination Some evidence that they are major contributors to adverse human health effects of DBPs Very little is known about N-DBPs Analytical chemistry is more complicated

109. Ranges of Org-N by types Estimates from literature surveys Classification50%ile90%ile99%ile DON3508002000 Free AA2050200 Combined AA40100400 Nucleic acids2050200 Amino Sugars40100400 Humic-N252001000 Others Order of magnitude estimates for organic nitrogen in surface waters (all values in µg-N/L)

110. Haloacetamides Mostly from HANs: N-Halogenated Forms Free (f) Combined (c ) Chlorine Sulfite Total (t) Measureable by GC

111. Ultrafiltration of DCAA and TCAA MW: DCAA, 128.9; TCAA, 163.4 Hua & Reckhow, 2007

112. Halamides Compounds – Monohaloacetamides Chloroacetamide, Bromoacetamide – Dihaloacetamides Dichloroacetamide (DCAD) Bromochloracetamide (BCAD) Dibromoacetamide (DBAD) – Trihaloacetamides trichloroacetamide & analogues Chlorination byproducts – Probably a bit less prevalent with chloramines – Pre-oxidation will probably reduce subsequent formation

113. Dichloroacetonitrile (DCAN) Surface Water Chemical Degradation in Distribution Systems

114. Amino Acids and Proteins Tyrosine Simple Amino Acids – some form THMs and HANs – Highest reactivity for activated AAs Tyrosine & Tryptophan: activated aromatic Cysteine: sulfhydryl group Proteins – many linked AAs; relatively unreactive polypeptide bonds – Reactions with proteins occurs most readily on AA side chains Alanine

116. Analysis of Organic N-chloramines Approach – Seems well suited to LC – Prior efforts with GC were not very successful e.g., tosyl derivatization Proposal – Fast analysis with UPLC – Parallel detection and analysis by Post-column reaction with I and absorbance LC/MS/MS

117. Some New DBPs Compound50%ile90%ile99%ile N-Chloroiminoacetonitrile0.30.62.5 N-N-Dichloroaminoacetonitrile0.61.45.5 N-Chlorophenylacetaldimine5.51353 Concentrations in µg/L Finished Drinking Water Concentrations