Tracing Nitrogenous Disinfection Byproducts after Medium Pressure

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Tracing Nitrogenous Disinfection Byproducts after Medium Pressure UV Water Treatment by Stable Isotope Labeling and High Resolution Mass Spectrometry Annemieke Kolkman,*,† Bram J. Martijn,‡ Dennis Vughs,† Kirsten A. Baken,† and Annemarie P. van Wezel†,§ †

KWR Watercycle Research Institute, P.O. Box 1072, 3430 BB Nieuwegein, The Netherlands PWN Water Supply Company North Holland, P.O. Box 2046, 1990 AA, Velserbroek, The Netherlands § Copernicus Institute of Sustainable Development, Utrecht University, Heidelberglaan 2, 3584 CS, Utrecht, The Netherlands ‡

S Supporting Information *

ABSTRACT: Advanced oxidation processes are important barriers for organic micropollutants (e.g., pharmaceuticals, pesticides) in (drinking) water treatment. Studies indicate that medium pressure (MP) UV/H2O2 treatment leads to a positive response in Ames mutagenicity tests, which is then removed after granulated activated carbon (GAC) filtration. The formed potentially mutagenic substances were hitherto not identified and may result from the reaction of photolysis products of nitrate with (photolysis products of) natural organic material (NOM). In this study we present an innovative approach to trace the formation of disinfection byproducts (DBPs) of MP UV water treatment, based on stable isotope labeled nitrate combined with high resolution mass spectrometry. It was shown that after MP UV treatment of artificial water containing NOM and nitrate, multiple nitrogen containing substances were formed. In total 84 N-DBPs were detected at individual concentrations between 1 to 135 ng/L bentazon-d6 equivalents, with a summed concentration of 1.2 μg/L bentazon-d6 equivalents. The chemical structures of three byproducts were confirmed. Screening for the 84 N-DBPs in water samples from a full-scale drinking water treatment plant based on MP UV/H2O2 treatment showed that 22 of the N-DBPs found in artificial water were also detected in real water samples.

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INTRODUCTION

Previous research has shown that application of medium pressure (MP) UV/H 2 O 2 treatment in drinking water production may lead to the formation of N-DBPs through nitrate photolysis. The complex mechanisms of nitrate photolysis involve the formation of various (intermediate) radicals, while the stable reaction product is nitrite.15,16 Experiments with organic molecules representative for specific constituents of natural organic matter show the ability to incorporate inorganic nitrogen in the organic matrix under the influence of MP UV irradiation,17−19 especially when aromatic low molecular weight compounds are involved. Thorn and Cox18 showed incorporation of inorganic nitrogen into aquatic NOM by UV irradiation, but no low-molecular weight nitrogen containing organics were identified.

Advanced oxidation processes, such as UV, UV/H2O2 and ozone, are gaining importance in (drinking) water treatment as barriers for organic micropollutants, such as pharmaceuticals and pesticides.1−3 These processes have however been shown to produce potentially harmful disinfection byproducts (DBPs). A vast amount of research has been performed to investigate the identities and occurrence of DBPs. Such studies are challenging, since each combination of disinfection method(s) and source water can generate a mixture of DBPs, and many factors affect the extent of DBP formation. More than 600 DBPs have been previously identified,4 and new research is identifying emerging DBPs such as nitrosamines and iodinated DBPs.5−8 Nitrogenous DBPs (N-DBPs) have gained attention only relatively recently.9−11 Toxicological information on these and other DBPs is often lacking or incomplete. Emerging brominated, iodinated, and nitrogenous DBPs seem to have a higher toxic potential than their chlorinated analogues.4,12−14 © 2015 American Chemical Society

Received: Revised: Accepted: Published: 4458

December March 10, March 11, March 11,

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epidemiology,34 food safety, and doping research.35 Moreover HR-MS enables chemical formula assignment and identification of unknown components.36 Using 15N labeled nitrate and LC HR-MS for artificial water samples treated with MP UV in laboratory studies, the first part of the current study shows that nitrogen originating from nitrate is indeed incorporated in N-DBPs. In total 84 newly formed N-DBPs are detected. The second part of the study evaluates the relevance of these results for a full-scale drinking water treatment plant. The fullscale drinking water treatment plant applies advanced oxidation and adds H2O2 prior to MP UV treatment. Water samples before and after MP UV treatment were screened for the 84 N-DBPs detected in the laboratory study. To indicate whether these N-DBPs may be responsible for the mutagenicity observed after advanced oxidation treatment, the Ames fluctuation test was performed in these samples as well. In addition, both analyses were performed at the end of the drinking water treatment process (i.e., after dune infiltration) to investigate the persistence of the N-DBPs and their potential biological activity during further treatment steps. This study offers innovative tools to monitor and control the presence of potentially hazardous DBPs. The results provide drinking water treatment utilities important insight into the types of N-DBPs formed, which will allow evaluation of their biological activity and assessment of public health risk.

Genotoxicity of treated drinking water is regularly evaluated by the Ames mutagenicity test. Effluents of MP UV/H2O2 drinking water treatment have been shown to induce a response in the Ames fluctuation test, indicating the presence of potentially mutagenic compounds.20−24 The genotoxic effect was shown to be effectively removed by post-treatment with granular activated carbon (GAC) filtration, which is applied to remove the excess of H2O2.23,24 Nitrate photolysis by MP UV irradiation in the presence of natural organic matter (NOM) was found to be the key parameter in the manifestation of an Ames test response after treatment of pretreated surface water, reconstituted water, and artificial water containing NOM and nitrate with MP UV technology.23,25 The formation of nitro- or nitroso aromatic compounds due to nitrate photolysis by MP UV irradiation in the presence of NOM is likely. Some nitro- and nitroso aromatic compounds are known to produce a response in the Ames test.9,26−28 The Ames test response observed after MP UV irradiation may be caused by multiple nitrated and nitrosated compounds at different concentrations. In order to quantify the total mutagenic effect of MP UV irradiation of water containing nitrate and NOM, preceding studies converted the Ames test response into 4-nitroquinoline oxide (4-NQO) equivalents, a known genotoxic compound commonly used as positive control in the Ames test. This showed that MP UV treatment of artificial water resulted in the formation of 120 ng/L 4-NQO equivalents in the water extracts.25,29 Since a Threshold of Toxicological Concern (TTC) of 10 ng/L has been derived for genotoxic substances in drinking water, only concentrations below this level are generally considered to pose a negligible human health risk. The relevance of these DBPs for public health should thus be further investigated. In addition, the TTC does not apply to high potency carcinogens, such as N-nitroso compounds, which need substance-specific risk evaluation.30 Substance-specific health risk assessment of DBPs can only be formed when identities and toxic potencies are known. Therefore, it is important to investigate which DBPs exactly are formed under the process conditions applied and what their contribution is to the mutagenic responses observed after MP UV water treatment. Insight into the chemical identity of DBPs not only is important for the evaluation of their health hazard but also is essential for the study of the behavior and fate of these products during drinking water treatment and may provide information on the chemical reactions involved in their formation. Studies investigating DBP formation processes using model compounds usually apply known precursors at much higher concentrations than present in full-scale water treatment plants. The first part of the present study therefore aims to unravel the identify the N-DBPs formed under the influence of MP UV irradiation at realistic NOM and nitrate concentrations. Based on the hypothesis that the formation of mutagenic compounds after MP UV water treatment results from the reaction of photolysis products from nitrate with (photolysis products of) NOM, we developed an innovative approach to detect these N-DBPs. The approach is based on labeling the newly formed N-DBPs with a stable isotope originating from 15N labeled nitrate. To detect the expected low concentrations of formed N-DBPs, concentration and sample pretreatment using solid phase extraction (SPE) and analysis by liquid chromatography coupled to high resolution mass spectrometry (LC HR-MS) was applied. HR-MS has proven to be excellently suited for screening samples for the presence of organic contaminants at low concentrations, e.g. in environmental analysis,31−33 sewer

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EXPERIMENTAL PROCEDURES - MATERIALS AND METHODS Chemicals. Pony Lake NOM was obtained from the International Humic Substances Society. Potassium nitrate (K14NO3) and 15N enriched (98% atom percentage enrichment) K15NO3 was obtained from Sigma-Aldrich. All solvents used were of analytical grade quality. Methanol (ultra resi-analyzed) and acetonitrile (ultra gradient HPLC grade) were purchased from Mallinckrodt Baker B.V. (Deventer, The Netherlands). SPE columns (200 mg Oasis HLB 6 mL glass cartridges) were supplied by Waters (Milford, MA, USA). Dimethyl sulfoxide (DMSO) (99.9%) was obtained from Acros Organics (Geel, Belgium). The compounds 4-nitrocatechol (3316-09-4) and 4-nitrophenol (100-02-7) were purchased from Sigma-Aldrich (Steinheim, Germany), and 2-methoxy-4,6-dinitrophenol (409763-6) was purchased from Vitas-M Laboratory (Moscow, Russia). Individual stock solutions for these compounds were prepared in methanol at a concentration of 100 mg/L. The internal standards atrazine-d5 and bentazon-d6 were purchased from CDN isotopes (Pointe-Claire, Canada) and Dr. Ehrenstorfer (Augsburg, Germany), respectively. Ultrapure water was obtained from a Milli-Q system (Millipore, Bedford, MA). Artificial Water Samples and MP UV Treatment. Artificial water consisted of ultrapure water, Pony Lake NOM (2.2 mg/L C), and nitrate (ca. 10.9 mg/L). Nitrate was added to ultrapure water as K14NO3, as the heavy stable isotope (K15NO3), and as a 1:1 mixture (14N/15N). Samples were treated with MP UV using a collimated beam (CB) setup. Concentrations of NOM and nitrate and experimental MP UV conditions were chosen at a level that positive responses in the Ames fluctuation test were expected, based on previous experiments,25 and thus potentially mutagenic compounds are presumably formed. Control samples were also prepared, i.e. artificial water containing NOM and 14N nitrate, which were not treated with MP UV. Fifty-five mL samples in a 60 × 35 mm crystallizing dishes were treated with MP UV using CB apparatus equipped with 4459

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compound identity was made. Confirmation of the identity was done by comparing the retention time, accurate mass, and fragmentation pattern to a reference standard, if available.36 Full-Scale Water Samples. Water samples for Ames testing and chemical screening for N-DBPs were taken from a full-scale water treatment facility (PWN, Heemskerk, The Netherlands) and the artificial dune recharge in March 2013. At this facility MP UV/H2O2 treatment is applied on conventionally pretreated surface water from Lake IJssel. The process conditions are 0.54 kWh/m3 and 6 mg H2O2/L. The UV installation is equipped with quartz sleeves, allowing utilization of the full emitted UV spectrum (200−300 nm). After MP UV/H2O2 treatment, GAC (EBCT, 9 min) is applied for H2O2 decomposition, followed by artificial dune water recharge via open infiltration channels. Dune water is reclaimed after at least 21 days by a closed abstraction well system and distributed as drinking water after aeration and rapid sand filtration. 1 L samples were taken before MP UV/H2O2 treatment, directly after MP UV/H2O2 treatment prior to GAC filtration, and in aerated reclaimed dune infiltration water (>21 days residence time). Table 1 gives the composition of these water samples.

a 3 kW medium pressure Hg lamp. The UV dose delivered to the solution was calculated using the UV dose calculation according to Bolton and Linden.37 UV intensity was measured using a radiometer with an unfiltered sensor (International Light Inc. (IL2000)). The irradiation path length was 19.5 mm. Multiple samples were irradiated under identical conditions in order to obtain sufficient sample volume. Sample Pretreatment and Analysis of Artificial Water Samples. Nine hundred mL of the water sample was acidified to pH 2.3 (HCl) and loaded over the SPE cartridge. Elution was performed with 7.5 mL of 8:2 (v/v) acetonitrile/methanol. The eluate was evaporated under a gentle stream of nitrogen until 250 μL. Next, 750 μL of ultrapure water containing the internal standards atrazine-d5 and bentazon-d6 was added to the extract. The final concentration of the internal standards atrazine-d5 and bentazon-d6 is 0.5 mg/L, corresponding to a concentration of 0.56 μg/L in the original sample. The extracts were analyzed using LC HR-MS. The LC system consisted of an Accela UHPLC system and Accela autosampler (Thermo Fisher Scientific, Bremen, Germany). The chromatographic separation was performed on a Xbridge C18 column (150 × 2.1 mm, 3.5 μm, Waters Corp.), and a 4.0 mm × 3.0 mm i.d. SecurityGuard C18 column (Phenomenex) was installed as precolumn. The gradient started with 5% acetonitrile, 95% water, and 0.05% formic acid (v/v/v), increased to 100% acetonitrile with 0.05% formic acid in 40 min, and subsequently was held constant for 10 min. The flow rate was 0.3 mL/min, and the column temperature was 21 °C. Ten μL of sample was used for injection. Detection of the extracts was performed using a hybrid LTQ-Orbitrap MS (Thermo Fisher Scientific, Bremen, Germany), using a Heated Electrospray (HESI) as ionization source. The source voltage was set at 3.0 kV and 2.5 kV for positive and negative ionization, respectively. The vaporizer and capillary temperature were 350 and 300 °C, respectively. Sheath, auxiliary, and sweep gas were set at 30, 10, and 10 arbitrary units, respectively. Full scan accurate mass spectra were acquired from 50 to 1300 Da at a resolution of 60,000 fwhm (at m/z 400). MS Data Analysis. The raw MS data files from the artificial water samples were analyzed using software tools (Sieve, Thermo Scientific) to detect differences between sets of triplicate samples from the 14N and 15N MP UV treated samples and the control samples. The intensity threshold for Sieve was set at 100.000 and 50.000 counts for the positive and negative ionization mode, respectively. The chromatographic data was compared from 2−40 min, the mass range from 50−1300 Da, and a frame time width of 2 min and a m/z width of 10 ppm. The triplicates from the MP UV treated 14N samples were compared with the triplicates from MP UV treated 15N samples, using the Sieve software to detect statistically significant differences. A two-tailed Student’s t test was used, and a p-value 21 days residence time)

DOC nitrate nitrite (mg C/L) (mg NO3−/L) (mg NO2−/L) 2.5 2.4 1.5

13.8 13.5 6.5

0.01 0.18 0.01

Bioassay and Chemical Analysis of Full-Scale Water Samples. The full-scale water samples were extracted according to the SPE method for Ames testing as described by Heringa et al.24 SPE eluates were dissolved in 100 μL of DMSO, yielding 10,000-fold concentrated extracts, which were not further diluted for application in the Ames test. The Ames fluctuation test, which is based on the same principle as the standard Ames assay,38,39 was applied according to Heringa et al.,24 including TA98 and TA100 strains (both with and without metabolic activation) obtained from Xenometrix GmbH (Allschwil, Switzerland) instead of TA98 and TAmix. Each sample was tested in triplicate. For chemical analysis, 20 μL DMSO extracts were diluted 20 times by adding 300 μL of ultrapure water and 80 μL of acetonitrile. Ten μL of this sample was analyzed on the HR-MS system as described above. The MS data was screened for the presence of the N-DBPs using Xcalibur software. Read-Across Analysis. Read-across was carried out with the aid of the OECD QSAR toolbox (version 3.2.0.103). In vitro mutagenicity (Ames gene mutation and the micronucleus assay), in vivo mutagenicity (Comet assay), and carcinogenicity in rat and mice of the selected compounds were assessed by obtaining measured data, if available. If not available, a category formation with analogues was carried out to perform read-across. Categories were based on DNA binding (Oasis), and subcategorization was carried out by eliminating aberrant organic functional groups or elements such as halogens.

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RESULTS AND DISCUSSION Labeling Strategy. Figure 1A depicts the strategy using stable isotopes to trace the formation of N-DBPs. When 15N is 4460

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Figure 1. A) Stable isotope labeling strategy for tracing N-DBPs. N-DBPs that are formed after MP UV when nitrate is present in its stable isotope labeled version, resulting in a peak in the mass spectrum with a Δm/z of 0.99704 compared to the normal N-DBP. Background ions will be present in both spectra with the same mass. B) Example of a N-DBP that is formed after MP UV treatment in artificial water samples. The EICs of m/z 238.0726 and m/z 239.0696 are shown (negative ionization mode), of which the elemental composition is C11H13O5N.

masses combined with a unique retention time representing N-DBPs were detected in the artificial water samples after MP UV treatment (see Supporting Information, Table 1). In most N-DBPs one nitrogen atom originating from nitrate was incorporated, while in 14 N-DBPs two nitrogen atoms were incorporated. 78 compounds were detected in negative ionization mode and 16 compounds in positive ionization mode. Ten compounds were detected with both ionization modes, resulting in a total of 84 unique N-DBPs. Concentrations of 1 up to 135 ng of bentazon-d6 equivalents (eq )/L for individual N-DBPs were found. The summed concentration after MP UV treatment of formed 84 N-DBPs is about 1.2 μg bentazon-d6 eq/L and 69 ng atrazine-d5 eq/L for negative and positive ionization, respectively. Some of the detected accurate masses occur at multiple retention times in the chromatograms (Figure 2). Multiple peaks are seen in the EIC of m/z 448.2675 and 449.2646 for the MP UV treated samples containing 14N-nitrate and 15N-nitrate, respectively, while these peaks are not detected in the sample without MP UV treatment. The EICs of the 14N and 15N N-DBPS seem to overlap. Most probably these are structurally related compounds, which elute at different retention times. Identification and Toxicological Evaluation of Detected N-DBPs. From the accurate mass, the most likely elemental composition of the N-DBPs can be deduced (Supporting Information, Table 1). The N-DBPs consist of carbon, nitrogen, oxygen, and sulfur atoms. The next step is identification and confirmation of the N-DBPs, which was conducted according to the criteria for identifying small molecules via HR-MS data as described by Schymanski et al.36 The identity of an N-DBP was confirmed (i.e., level 1)36 by analyzing reference standards when available and matching the

incorporated into a newly formed N-DBP, the accurate mass spectrum shows a mass difference of 0.99704 Da compared to the same N-DBP formed in the presence of normal nitrate. This mass difference is used to screen the raw data files for N-DBPs. Background ions do not show this characteristic mass difference and can therefore be distinguished from N-DBPs. Figure 1B as example shows extracted ion chromatograms (EICs) from an N-DBP with the elemental composition C11H13O5N. For the 14N nitrate MP UV treated sample a chromatographic peak is seen, for the EIC of m/z 238.0726, while this peak was not present in the same sample without MP UV treatment. In the 15N nitrate MP UV treated sample a chromatographic peak is seen at the EIC of m/z 239.0696. In the sample in which a 1:1 mixture of normal and 15N labeled nitrate is added, both peaks are seen. Both versions of the N-DBP coelute, which indicate that the 15N atom does not influence retention time. Both these peaks are not present in the sample without MP UV treatment. The 1:1 mixture sample can be used to search for peak pairs with a fixed mass difference and same retention time, to make data analysis more straightforward. At the time of analysis appropriate software tools were not yet available in our laboratory, and we used the 1:1 samples as a control to check if both the 14N and 15 N version of a N-DBP are formed after MP UV. These results demonstrate the incorporation of a nitrogen atom originating from nitrate into the organic matrix (NOM), supporting the hypothesis that nitrate photolysis is involved in the formation of N-DBP. The isotope labeling strategy enables us to trace N-DBPs in a highly sophisticated and sensitive manner. Detected N-DBPs in Artificial Water Samples. The approach resulted in a list of masses that are statistically different between the 14N and 15N prepared samples. In total 94 accurate 4461

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Figure 2. Elution of structural isomers. A) The EIC of m/z 448.2675 (negative ionization mode, elemental composition C20H39O8N3) is shown of the untreated 14N nitrate sample. B) The EIC of m/z 448.2675 is shown of the MP UV treated samples containing 14N nitrate. C) The EIC of m/z 449.2646 is shown (negative ionization mode, elemental composition C20H39O8N215N), of the MP UV treated samples containing 15N nitrate.

experimental data are considered insufficient to conclude on the genotoxicity of this compound.40,41 In silico read across prediction shows that based on its chemical structure, 2-methoxy-4,6-dinitrophenol is expected to yield a positive response in the Ames test with and without metabolic activation. There are insufficient experimental data available for the three identified N-DBPs and their analogues to make predictions for other end points related to genotoxicity and carcinogenicity. The selected compounds do have a structural alert for DNA binding, suggesting that indirect DNA damage through adduct formation may be possible. It has been reported that nitration of NOM, in particular its aromatic substituents, may promote halonitroalkane formation upon postchlorination or chloramination. Chlorination of drinking water upstream of MP UV treatment was shown to increase halonitromethane and chloropicrin formation.11 Since these substances are known toxicants,42 the N-DBPs that were shown to be formed in the present study may result in DBPs that are of human health concerns when they are not removed by granular activated carbon and MP UV drinking water treatment is followed by chlorination or chloramination. Screening for N-DBPs and Mutagenicity in Full-Scale Water Samples. The 84 N-DBPs that were detected in the artificial water samples were screened for by HR-MS in water samples from a full-scale drinking water treatment plant, i.e. before and after MP UV/H2O2 treatment and after dune infiltration. These samples were also analyzed using Ames fluctuation tests. Of the 84 N-DBPs discovered in the artificial water samples, in total 22 N-DBPs were also detected in the full-scale MP UV treatment samples treatment (see Supporting Information, Table 2). The three N-DBPs whose identities were confirmed, namely 4-nitrophenol, 4-nitrocatechol, and 2-methoxy-4,6-dinitrophenol, were also present in treated full-scale water samples. These findings

mass, retention time, and MS/MS fragmentation patterns of the reference standard with the N-DBP in the sample. A pragmatic approach was applied for buying the reference standards, based on availability, rationale, and costs. The identities of 4-nitrophenol, 4-nitrocatechol, and 2-methoxy-4,6-dinitrophenol (Figure 3) were confirmed (see Supporting Information,

Figure 3. Structures of 3 N-DBPs of which the identity was confirmed. From left to right: 4-nitrophenol, 4-nitrocatechol, and 2-methoxy-4,6dinitrophenol.

Figures S1−S3). Nitrophenols and nitrocatechols were identified as reaction products after UV irradiation of dissolved nitrate in previous research as well.19,25 There is an indication that 2-hydroxy-5-nitrobenzoic acid, 5-nitrovanillin, 4-nitrophthalic acid, nitrobenzenesulfonic acid, and 2,4-dinitrophenol are formed after MP UV treatment; however, the lack of a good MS2 spectrum of the N-DBP in the artificial sample, or a slightly different retention time, makes the evidence not 100% (see Supporting Information, Table 3). Toxicological evaluations of the identified N-DBPs 4-nitrocatechol, 2-methoxy-4,6-dinitrophenol, and 4-nitrophenol are not available or very limited. Although 4-nitrophenol generally does not appear to be genotoxic in vitro e.g. in the Ames test, it may cause chromosomal aberrations in vitro. No data have been reported regarding the carcinogenic potential of 4-nitrophenol in humans, and results of animal experiments are equivocal. Hence, 4462

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Figure 4. A) Ames fluctuation test response (bars denote average values, error bars indicate standard deviations (n = 3); concentration factor is 10,000) and B) broad chemical screening data (HR-MS, summed response of 22 N-DBPs detected in eq/L) of water samples in a full-scale drinking water production chain. Samples were obtained (i) before MP UV/H2O2 treatment, (ii) after MP UV/H2O2 treatment, and (iii) after dune infiltration. For the Ames fluctuation test, a negative control is included.

will have its own potency in the Ames fluctuation test. In addition, the intensity of each compound in the MS is dependent on ionization efficiency. Besides, both the bioassay and chemical measurements yield semiquantitative results. Therefore, the correlation between the concentration of substances expressed in equivalents and the mutagenic response is not statistically tested. Further identification of the detected N-DBPs, using effect directed analysis45 to pinpoint the source of the mutagenicity or individual testing of these substances in Ames tests, will provide more insight into the relation of the N-DBPs with the observed mutagenicity. The innovative approach to identify DBPs formed during advanced oxidation processes presented here is an important step forward to elucidate the mechanisms of DBP formation, their behavior during drinking water treatment processes, and their potential human health hazard evaluation. Further research is needed to investigate whether the same DBPs are formed when different organic matter compositions or different drinking water treatment conditions are in place.

link the results obtained in a MP UV laboratory setting to a full-scale drinking water treatment plant and show that N-DBPs formed in a laboratory setting are relevant for fullscale MP UV/H2O2 processes. The summed concentration (eq/L) of the 22 N-DBPs in the full-scale drinking water treatment setting, with 82 ng bentazond6 eq/L, are a factor of 9 lower than the summed concentration of the same 22 N-DBPs found in the artificial water samples. There are several explanations for this and for the absence of 62 of the N-DBPs in the full-scale samples above detection limit. The exact type of NOM present determines the exact DBPs formed.43 So in the full-scale samples several different N-DBPs other than the 84 which were screened for might be formed, due to differences in NOM characteristics. Further research should elucidate which NOM components specifically serve as precursors for the N-DBPs, and whether the same N-DBPs are formed in water with different composition of organic material. The presence of algal organic matter or anthropogenic pollutants in the surface water used for drinking water production may give rise to formation of additional DBPs which are not screened for in this study. For the full-scale samples, not only MP UV but also H2O2 was applied. H2O2 decomposition may oxidize part of the N-DBPs formed, explaining why a part of the N-DBPs is not detected or detected at a lower concentration in the full-scale samples.22,44 Figure 4 compares the Ames fluctuation test response (4A) and the results of the chemical screening for the N-DBPs (4B) in the full-scale drinking water treatment samples. The results indicate an increased response in both the Ames fluctuation test and the chemical analysis (expressed as summed equivalent concentrations) after MP UV/H2O2 treatment and a subsequent decrease in both these responses after dune infiltration albeit not exactly of the same order of magnitude. The increase in response after MP UV/H2O2 treatment is more pronounced in the TA98 strain than in the TA100 strain. This may indicate that substances causing frameshift mutations are formed. The similar trends of the chemical screening results and the mutagenic response indicate that (part of) the 22 N-DBPs present in the full scale water may contribute to the observed mutagenicity. The mutagenic potency is as yet unknown for the majority of these N-DBPs. The detected Ames fluctuation test response represents the total effect of a mixture of mutagenic (such as 2-methoxy-4,6dinitrophenol) and nonmutagenic (such as 4-nitrophenol and 4-nitrocatechol) N-DBPs. Each mutagen in the mixture of DBPs

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ASSOCIATED CONTENT

S Supporting Information *

Supplementary Tables 1−3 and Figures S1−S3. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Phone: 31 030 60 69655. Fax: 31 030 60 61 165. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This study was performed within the framework of the Joint Research Program of the Dutch water companies (BTO). Merijn Schriks is acknowledged for performing chemical read across analysis and Roberta Hofman-Caris for sharing her expertise and for her critical review of this document. We are grateful to Moayad Aljammaz from Wageningen University Division of Toxicology for performing the UV collimated beam experiments.

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REFERENCES

(1) Lekkerkerker-Teunissen, K.; Benotti, M. J.; Snyder, S. A.; van Dijk, H. C. Transformation of atrazine, carbamazepine, diclofenac and

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Environmental Science & Technology

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DOI: 10.1021/es506063h Environ. Sci. Technol. 2015, 49, 4458−4465

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DOI: 10.1021/es506063h Environ. Sci. Technol. 2015, 49, 4458−4465