Application of effect-directed analysis to identify mutagenic nitrogenous disinfection by-products of advanced oxidation drinking water treatment

Transcription

1 Application of effect-directed analysis to identify mutagenic nitrogenous disinfection by-products of advanced oxidation drinking water treatment Kirsten Baken, Dennis Vughs, Annemieke Kolkman Bram Martijn

2 Chemical analysis and bioassays in the drinking water production train Surface water is treated to produce drinking water Disinfection (chlorination, ozonation, UV radiation) Removal of micropollutants (adsorption, membranes, advanced oxidation) Advanced oxidation processes (AOP) involving UV and ozone effectively remove persistent pathogens and polar chemical contaminants (e.g. pharmaceuticals, pesticides) AOP may generate mixtures of disinfection by-products (DBPs) More than 600 (chlorinated, iodinated, brominated, nitrogenous) DBPs have been identified but many more are formed Chemical identity and toxicity are often not known Ames fluctuation test detects the presence of unknown, potentially mutagenic N-DBPs Genotoxicity has been observed in water treated by medium pressure (MP) UV/H 2 O 2

3 Mutagenic byproducts of MP UV treatment Heringa et al., 2011

4 4 Role of Natural Organic Matter (NOM) and nitrate TA98-S9 cf 20,000 IHSS Pony Lake NOM IHSS Pony Lake NOM with nitrate positive wells method control positive control blank MP UV/H2O2 From PWN technologies Nitrate photolysis by MP UV irradiation in the presence of NOM was found to be the key parameter in the manifestation of an Ames test response; nitro radicals as (reactive) intermediates? Martijn et al., 2014

5 Identification of disinfection by-products Aim of the study Provide information on the chemical reactions and process conditions involved in their formation Study of the behavior and fate during drinking water treatment Perform human health risk assessment

6 Development of an innovative tool for the detection of N-DBPs of MP UV treatment Nitrogen labeling principle 6 NOM + nitrate (NO 3- ) + MP UV nitrogen containing by-products NOM + 14 NO 3- + MP UV nitrogen containing by-products NOM + 15 NO 3- + MP UV nitrogen containing by-products Isotope tagging in the mass spectrometer Kolkman et al., 2015

7 7 Results 84 N-DBPs detected Negative mode 78 detected compounds 54 different chemical formulas 14 compounds with 2x 15 N label Total concentration = 1234 ng/l (bentazon-d6) Positive mode 16 detected compounds 6 different chemical formulas 0 compounds with 2x 15 N label Total concentration = 69 ng/l (atrazin-d5) 6 compounds detected only in positive mode Kolkman et al., 2015

8 8 Full scale water treatment Results bioassays versus chemical analysis Results Ames fluctuation test Results Orbitrap analysis (neg) Positive wells 16,0 14,0 12,0 10,0 8,0 6,0 6,7 7,7 12,3 14,0 4,7 5,2 TA98-S9 TA98+S9 ISTD equivalents (ng/l) 90,0 80,0 70,0 60,0 50,0 40,0 30,0 81,9 4,0 2,0 0,0 Before UV After UV After dune infiltration 20,0 10,0 0,0 10,0 0,8 Before UV After UV After dune infiltation Kolkman et al., 2015

9 Dennis Vughs Non Target Results Genotoxic potential of identified N-DBPs Calculation of the chemical formula Compound CAS nr Formula Proposal for structural formula Analysis of samples and reference standards Confirmation of by-products (matching retention time and MS/MS spectrum) Genotoxic potential (based on measured data and/or QSAR analysis) 4-nitrophenol C 6 H 5 NO Overall evidence points to absence of mutagenicity in Ames test; insufficient data to assess other 3 genotoxicity and carcinogenic potential. 4-nitrocatechol C 6 H 5 NO Probably not mutagenic in Ames test; insufficient data to assess other genotoxicity and 4 carcinogenic potential. 4-nitro-1,3-benzenediol C 6 H 5 NO Structure suggests genotoxic potential. 4 2-nitrohydroquinone C 6 H 5 NO 4 Structure suggests genotoxic potential. 2-hydroxy-5-nitrobenzoic acid C 7 H 5 NO 5 Structure suggests genotoxic potential but no mutagenicity. 4-hydroxy-3-nitrobenzoic acid C 7 H 5 NO 5 Structure suggests genotoxic potential. 2-hydroxy-3-nitrobenzoic acid C 7 H 5 NO 5 Structure suggests genotoxic potential. 2,4-dinitrophenol C 6 H 4 N 2 O 5 Weight-of-evidence indicates no mutagenicity and genotoxicity, but clastogenicity and carcinogenicity cannot be excluded. 5-nitrovanillin C 8 H 7 NO 5 Structure suggests genotoxic potential but no mutagenicity. 4-nitrobenzenesulfonic acid C 6 H 5 NO 5 S Mutagenicity and genotoxicity are not expected. 4-nitrophthalic acid C 8 H 5 NO 6 Structure suggests genotoxic potential. 2-methoxy-4,6-dinitrophenol C 7 H 6 N 2 O 6 Potentially mutagenic in Ames test; insufficient data to assess other genotoxicity and carcinogenic potential. 3,5-dinitrosalicylic acid C 7 H 4 N 2 O 7 Structure suggests genotoxic potential. dinoterb C 10 H 12 O 5 N 2 Structure suggests genotoxic potential. Kolkman et al., 2015

10 10 Effect directed analysis approach Aim of the study Genotoxic potential of the identified N-DBPs does not explain the observed Ames response Application of effect directed analysis to identify mutagenic nitrogenous disinfection byproducts

11 MP UV treatment SPE extraction 3 ml extract 30 L Artificial water 15 L treated sample 15 L untreated sample SPE extraction Procedure equal to treated water sample NORMAN, April 11th 2017 Experimental design 11 1 ml extract 1 ml extract fractionation by preparative HPLC-UV Fraction Fraction Fraction Fraction Fraction Fraction Fraction Fraction evaporation 120 µl DMSO 120 µl DMSO (8x) Ames test Ames test LC-Orbitrap MS analysis Vughs et al., 2015

12 12 Fractionation and concentration of water extracts The total concentration of byproducts detected in the fractionated samples was in agreement with the total concentration detected in the unfractionated samples The majority of the N-DBPs were shown to be predominantly present in one of the fractions Untreated Treated Concentration (ng/l ISTD) Fractions (summed) Unfractionated sample Vughs et al., 2015

13 13 N-DBPs in fractionated water extracts Positive wells # Identity and mutagenicity TA98-S9 untreated TA98+S9 untreated TA98-S9 treated TA98+S9 treated 250 Concentration (ng/l ISTD) Untreated Treated NC PC1 PC2 Untreated fraction 1 MP UV treated fraction 1 Untreated fraction 2 MP UV treated fraction 2 Untreated fraction 3 MP UV treated fraction 3 Untreated fraction 4 MP UV treated fraction 4 Untreated fraction 5 MP UV treated fraction 5 Untreated fraction 6 MP UV treated fraction 6 Untreated fraction 7 MP UV treated fraction 7 Untreated fraction 8 MP UV treated fraction 8 Vughs et al., 2015

14 14 Top 5 of N-DBPs per fraction Mass Conc. Formula Compound (m/z) (ng/l) Fraction (1) (1) (1) 0.8 C 6 H 5 O 4 N 4-nitrocatechol (1) 0.7 C 8 H 5 O 6 N 4-nitrophthalic acid (2) 0.4 Fraction (2) 42.2 C 7 H 5 O 5 N 4-hydroxy-3-nitrobenzoic acid C 6 H 5 O 3 N 4-nitrophenol (1) 26.2 C 6 H 5 O 4 N 4-nitrocatechol (2) (2) 10.0 Fraction (1) 34.9 C 14 H 23 O 7 N C 9 H 7 O 5 N (2) (2) 7.4 C 8 H 5 O 7 N C 7 H 6 O 6 N 2 2-methoxy-4,6-dinitrophenol Mass Conc. Formula Compound (m/z) (ng/l) Fraction C 7 H 6 O 6 N 2 2-methoxy-4,6-dinitrophenol (3) 11.7 C 14 H 23 O 7 N C 11 H 13 O 5 N (1) (1) 8.3 C 14 H 23 O 7 N Fraction C 8 H 7 O 6 N Structural isomer of 5-hydroxy-4- methoxy-2-nitrobenzoic acid C 13 H 17 O 5 N C 10 H 12 O 5 N 2 dinoterb C 7 H 4 O 7 N 2 3,5-dinitrosalicylic acid Fraction (3) 56.2 C 7 H 5 O 5 N 2-hydroxy-5-nitrobenzoic acid C 7 H 4 O 7 N 2 3,5-dinitrosalicylic acid (3) C 10 H 12 O 5 N 2 dinoterb Based on (predicted) genotoxic potential 4-nitrophthalic acid, 4-hydroxy-3-nitrobenzoic acid, 2- methoxy-4,6-dinitrophenol, dinoterb and 3,5-dinitrosalicylic acid may have contributed to the observed mutagenicity. Vughs et al., 2015

15 Mass (m/z) Which N-DBPs explain mutagenicity 50 in fraction 7 and 8? RT (min) Mode fraction Conc. (ng/l) Formula (1) pos C 16 H 21 O 7 N (2) pos C 16 H 21 O 7 N ID Positive wells # NORMAN, April 11th TA98-S9 untreated TA98+S9 untreated TA98-S9 treated TA98+S9 treated (3) pos C 16 H 21 O 7 N neg C 10 H 12 O 5 N 2 Dinoterb neg 8 2.1? NC PC1 PC2 Untreated fraction 1 MP UV treated fraction 1 Untreated fraction 2 MP UV treated fraction 2 Untreated fraction 3 MP UV treated fraction 3 Untreated fraction 4 MP UV treated fraction 4 Untreated fraction 5 MP UV treated fraction 5 Untreated fraction 6 MP UV treated fraction 6 Untreated fraction 7 MP UV treated fraction 7 Untreated fraction 8 MP UV treated fraction 8 Concentration (ng/l ISTD) Untreated Treated Vughs et al., 2015

16 16 Conclusions Nitrogen labeling is a new innovative approach for the detection of nitrogen containing by-products By applying a fractionation method to MP UV treated water samples, the presence of N-DBPs and mutagenicity in the Ames test were shown to be correlated A selection of byproducts that are likely to contribute to the mutagenic response were identified Outlook Testing of (mixtures of) the N-DBPs in the Ames fluctuation tests Identification and quantification of additional by-products Relevance for full-scale treatment and varying process conditions (water composition, AOP conditions) Refinement of methodology (number of fractions, bioassay panel)

17 Bridging science to practice Thanks for your attention! This study was performed within the framework of the Joint Research Program of the Dutch water companies (BTO) and was co-financed with TKI-funding from the Topconsortia for Knowledge & Innovation (TKI s) of the Ministry of Economic Affairs of the Netherlands. 17

18 18 Bridging science to practice More articles and images in our online annual report annualreport.kwrwater.nl KWR Watercycle Research Institute