Identification of Mutagenic Aromatic Amines in River Samples with

Article pubs.acs.org/est

Identification of Mutagenic Aromatic Amines in River Samples with Industrial Wastewater Impact Melis Muz,*,†,‡ Janek Paul Dann,† Felix Jag̈ er,§ Werner Brack,†,‡ and Martin Krauss† †

Department Effect-Directed Analysis, Helmholtz Centre for Environmental Research - UFZ, Permoserstrasse 15, 04318 Leipzig, Germany ‡ Department of Ecosystem Analyses, Institute for Environmental Research, RWTH Aachen University, Worringerweg 1, 52074 Aachen, Germany § Synchem UG & Co. KG, Am Kies 2, 34587 Felsberg-Altenburg, Germany S Supporting Information *

ABSTRACT: Aromatic amines are one of the most important classes of compounds contributing to surface water mutagenicity due to their widespread occurrence as precursors and transformation products of dyes, pharmaceuticals, agrochemicals, and other compound classes. In this study, we implemented a workflow including novel analytical and data evaluation methods aiming to identify aromatic amines in six mutagenic wastewater effluents from a chemical-industrial area in Germany, collected by the passive sampler Blue Rayon. We identified 14 amines including the two potent mutagenic aromatic amines 2,3- and 2,8-phenazinediamine, which were reported for the first time as environmental contaminants. These two isomers accounted between 4.2 and 86% of the mutagenicity of the blue rayon extracts and may be byproducts of dye production at the studied site.

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INTRODUCTION Treated wastewater effluents are one of the most important sources of emerging contaminants in European surface waters.1,2 Conventional treatment systems are inadequate to remove many of these compounds completely.3,4 Hence, mixtures of emerging contaminants including pharmaceuticals, illicit drugs, industrial chemicals, personal care products, and their transformation products are discharged into rivers, lakes, and coastal waters with a broad range of concentrations. These chemicals might impact on the aquatic environment by exhibiting sublethal or even lethal effects on organisms. Mutagenicity/genotoxicity is a frequently observed sublethal effect caused by wastewater effluents5−7 that might compromise safe drinking water production and the fitness and the genetic structure of aquatic wildlife populations.8 Therefore, mutagens should be thoroughly examined in terms of their source, fate, and potencies. The large industrialized area of Bitterfeld (Germany) is a known hot spot of chemical legacy pollution9,10 and is still one of the most important chemical industry sites in Germany. It continues to contribute to the pollution of the rivers Mulde and Elbe11−13 which poses a considerable threat to human health since River Elbe is a source of drinking water via river bank filtration and a water supply for agriculture in Central Europe.14−16 In a previous study Hug et al.11 investigated the causative chemicals in six mutagenic wastewater effluents of a treatment plant in Bitterfeld. Although peaks covarying with mutagenicity could be discriminated from the bulk of chemical © 2017 American Chemical Society

signals and characterized, no individual mutagens were identified to explain the observed effect by the effluents. The results indicated that the fraction of nitrogen containing compounds was 30 times higher in the subset of peaks covarying with mutagenicity than in the whole set of chemical signals suggesting compounds with a nitro group or mutagenic aromatic amines as relevant candidates. An increase of mutagenicity after metabolic activation by S9 in the Ames fluctuation assay suggested that aromatic amines might be main contributors to this effect.17 Aromatic amines are abundant in effluents due to their utilization as precursors in pesticides, dyes, and pharmaceuticals.18−21 However, none of the suspected mutagenic aromatic amines were detected in the samples. Therefore, we implemented a workflow aiming to identify unknown mutagenic aromatic amines in surface waters and applied it to the river samples bearing likely mutagenic amines based on previous findings.11 In the present study Blue Rayon (BR) extracts were used, which were collected in parallel to the water samples studied earlier.11 BR is a fabric that facilitates the adsorption of polycyclic planar structures and is used successfully for the identification of novel mutagenic 2-phenylbenzotriazoles22,23 and mutagenic heterocyclic amines like 2-amino-1-methyl-6Received: Revised: Accepted: Published: 4681

January 24, 2017 March 27, 2017 April 3, 2017 April 7, 2017 DOI: 10.1021/acs.est.7b00426 Environ. Sci. Technol. 2017, 51, 4681−4688

Article

Environmental Science & Technology

scan data was carried out by MZmine 2.10,31 and peak lists of derivatized and underivatized samples were used to create a data matrix for each sample to find the parent-derivative matches based on a mass difference of 163.0018 ± 0.0008 (±5 ppm) corresponding to the addition of an NBD-F molecule. Details of the settings for LC-HRMS analysis and MZmine are given in the SI, sections S1.2 and S1.3. HRMS/MS information on the derivatized and underivatized samples was acquired at the same LC and MS conditions but with an additional data dependent scan (nominal resolving power of 35,000 at m/z 200) containing an inclusion list of analyte and derivative masses. Derivatives were confirmed with 1−8 diagnostic fragments observed in the MS/MS spectra. Molecular formulas of the analyte peaks were manually assigned using the XCalibur software based on measured accurate masses and isotope patterns and searched in the Chemspider database. Formulas resulting in no structure bearing an aromatic ring were removed from further evaluation. MS/MS spectra of analytes with confirmed derivatives were used to generate candidate lists by MetFrag 2.2 (command line version).32 Details of the settings for MetFrag are given in the SI, section S1.4. To limit the number of candidates to a manageable size, a score of 0.7 was selected as cutoff, and the candidates above this score were further reduced to those with a number of ChemSpider references ≥10 as a proxy for the commercial importance and environmental relevance. Spectral information from MassBank33 was used, when available, to support the candidate selection process. Candidate Selection. Two additional methods were applied with the underivatized samples to reduce the number of candidates remaining after the MetFrag score and reference number cutoffs. pH-Dependent LC Retention. The retention of ionizable compounds depends strongly on the pH of the mobile phases in a reversed phase (RP)-LC system.34 Most aromatic amines have pK a values between 2.6 and 5.5 resulting in a predominantly protonated state at pH values below 2.6 and a neutral state at pH values higher than 5.5. This phenomenon results in different retention times of aromatic amines with mobile phases having different pH values and can be used for candidate selection, as shown previously in ref 35. Therefore, aliquots of underivatized BR extracts were separated using the same gradient elution program with different mobile phase combinations at pH 2.6, 6.4, and 10. An aromatic amine should have the lowest RT at pH 2.6 and higher but similar RTs at pH 6.4 and 10, whereas for aliphatic amines having pKa values higher than 8, RTs at pH 2.6 and 6.4 should be similar and a higher RT at pH 10 should be observed. pKa values of the candidates were calculated with Jchem for Excel (version 6.3.0),36 and the details of the method are given in the SI, section S1.5. Hydrogen−Deuterium Exchange (HDX). HDX provides information on the number of polar functional groups via counting the exchangeable hydrogen atoms connected to heteroatoms. For HDX experiments, the same gradient elution program was used but with deuterium oxide (99.9 atom-% D, Sigma-Aldrich) containing 0.1% formic acid as eluent A and methan-d1-ol (99.8 atom-% D, Sigma-Aldrich) as eluent B, and full scan chromatograms were recorded with the same method. Compound Confirmation. For the remaining candidates that satisfy the criteria of both methods, a reference standard for the structures with the highest number of references in ChemSpider was obtained when available. Compounds

phenylimidazo(4,5-b)pyridine (PhIP), 1,4-dimethyl-5H-pyrido[4,3-b]indol-3-amine (Trp-1), and 3,8-dimethyl-3H-imidazo[4,5-f ]quinoxalin-2-amine (MeIQx) in different media such as water,24 urine,25,26 and grilled/cooked meat.27 Using BR extracts allowed for a more focused search for mutagenic aromatic amines eliminating most of the aliphatic amines which are known to be nonmutagenic. Furthermore, this approach allows for the concentration of aromatic compounds (including aromatic amines) above analytical detection levels in surface waters. A selective detection of aromatic amines in the complex mixture of aromatic compounds collected with BR was achieved by using a workflow that includes a previously developed derivatization method28 to label compounds containing amino groups and/or heterocyclic nitrogen atoms and two complementary LC approaches. Candidates were confirmed with reference standards when available, and the ones correlating with the mutagenicity of the water samples were tested with the Ames fluctuation assay (Ames II test) with metabolic activation by S9 to evaluate their mutagenic potencies and their contribution to the observed mutagenicity of the BR extracts.

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MATERIALS AND METHODS Details of the reagents used are given in the Supporting Information (SI), section S1.1. Sample Collection and Preparation. The WWTP of Bitterfeld-Wolfen receives domestic and industrial wastewater from the municipality and the large industrial area of Bitterfeld/ Wolfen. The industrial park contains 30 companies producing a wide variety of chemicals including intermediates, pharmaceuticals, and dyes. The samples were collected in 2011 from River Mulde, a tributary of the River Elbe, at the location where the effluent of the WWTP is discharged into the river. In total six samples were collected in consecutive weeks in parallel with the grab water samples of the previous study.11 Two batches of 5 g of BR (in total 10 g) were prepared and hung out in the river as described by Sakamoto and Hayatsu et al.29 and exposed for 24 h. The exposed BR was then collected and extracted according to Kummrow et al.30 Briefly, the exposed BR was rinsed with bidistilled water to remove the particles. The BR was agitated with a 160 mL solution of methanol/ammonium hydroxide (50:1 v:v) per g BR. This process was conducted twice, and the solutions were combined and evaporated to dryness. The extracts were then redissolved in 5 mL of methanol resulting in a concentration of 2 g BR mL−1 and stored in −20 °C in glass vials until further analysis. Derivatization. Aliquots of 30 μL were taken from each BR extract and derivatized with 4-fluoro-7-nitrobenzooxidazole (NBD-F) as reported previously28 by adding 10 mM of NBD-F (10 μL) and 20 mM of ammonium acetate buffer at pH 5.7 (2 μL) and heating the mixture to 80 °C for 30 min. LC-HRMS Measurement and Data Evaluation. Liquid chromatography separation of the derivatized and underivatized aliquots was achieved with a Thermo Ultimate 3000 LC system equipped with a phenyl-hexyl column (Accucore PhenylHexyl 150 mm × 3 mm, 2.6 μm particle size, Thermo) using a gradient elution with 0.1% formic acid and methanol with a flow rate of 0.2 mL min−1. For the initial analysis only full scan acquisition was performed by a QExactive Plus instrument (Thermo) with a nominal resolving power of 140,000 (at m/z 200) and mass accuracy BR6 (68.6) > BR4 (53.3) > BR2 (47.5) > BR1 (24) > BR3 (9.6) unlike the order of the grab samples tested in the previous study of Hug et al.11 The concentration response curves for all BR extracts are given in Figure 2.

produced/observed in Bitterfeld according to the suspect list compiled by Hug et al.37 and fitting the criteria above were obtained as reference standards independent of the number of references. The standard of 2,8-phenazinediamine was synthesized as described in the SI, section S1.6. The standards were derivatized with NBD-F, and both derivatized and underivatized peak RT and MS/MS information were used for confirmation. The level of confidence classification by Schymanski et al.38 was used to describe the confirmation levels of the identified compounds. Mutagenicity Tests. The mutagenicity of the confirmed compounds and BR extracts was tested with the Ames fluctuation assay (Ames II test) using strain TA98 with metabolic activation by S9. The mutagenic potencies were evaluated by fitting the results to the exponential equation reaching a maximum of 48, and the first derivative at a concentration of 0 representing the slope of the linear part of the concentration−response curves was used for the quantitative assessment of mutagenicity and given as number of revertants/mg BR equivalent. Details are given in the SI, section S1.7. The summary of the applied workflow for identification of possible aromatic amine mutagens is given in Figure 1.

Figure 2. Concentration−response plots of BR extracts (strain TA98 in the presence of S9).

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Peak Selection. Data matrices were created for each extract with the peak lists of the derivatized and underivatized samples obtained from MZmine peak detection of the LC-HRMS analyses. Based on the search for the mass difference of an NBD-F molecule (163.0018 ± 5 ppm), 130 potential parentderivative matches were found in total in the six extracts. More than half of the matches were detected in more than one extract indicating a continuous input of these compounds to the river. In total, 89 derivatives were confirmed with 2−8 diagnostic

RESULTS AND DISCUSSION Mutagenicity of BR Extracts. The BR extracts were tested with strain TA 98 in the presence of S9, as 1.2 mg of BR equivalent per well, being the highest concentration. Mutagenic activities of the extracts were evaluated by the slopes (a*b) calculated according to eq S1. The order of mutagenicity as given in a number of revertants/mg BR equivalent in brackets 4683

DOI: 10.1021/acs.est.7b00426 Environ. Sci. Technol. 2017, 51, 4681−4688

a

0.01−0.21 0.03−0.52

C9H7NO

C9H7NO

C5H5N5O C9H11NO2 C9H11NO3

C11H12N2O2

C12H10N4

C12H10N4

146.0599 (−2.5)

146.0599 (−2.6)

152.0565 (−1.0) 166.0862 (−0.5) 182.0811 (−0.4)

205.0970 (−0.6)

211.0977 (−0.8)

211.0977 (−1.1)

4684

0.09−0.65

C14H19N5O2

C33H30N4O2

290.1607 (−1.4)

515.2444 (+0.4)

pH-dependent LC retention method not applicable.

0.001−0.35

C9H15N3O2S C16H21NO2

230.0956 (−0.8) 260.1643 (−0.6) 0.01−0.39 0.02−0.09

0.01−0.40

0.05−0.52 0.03−3.04 0.15−3.26

0.001−0.01

0.001−0.02

0.01−0.05

C9H7NO

146.0599 (−2.5)

0.02−1.30

detected concn range in the BR extracts (mg L‑1)

C7H10N2

neutral formula

123.0918 (+1.1)

detected protonated mass [M + H]+ (mass accuracy in ppm)

9 (1)

957(31)

186 (10) 191 (6)

71 (39)

71 (39)

531 (40)

85 (21) 252 (87) 301 (58)

148 (47)

44 (21)

179 (56)

283 (85)

120 (9)

a

5 (1)

810 (9)

26(2) 160 (6)

57 (30)

6 (1)

173 (2)

87 (4) 74 (5)

12 (9)

12 (9)

23 (11) 76 (44) 114 (30)

a 78 (38) a

57 (30)

37 (25)

33 (19)

29 (28)

33 (3)

no. of candidates complying with HDX (ref > 10)

100 (34)

26 (10)

51 (41)

143 (55)

no. of candidates no. of candidates complying with a MetFrag score with pH-dependent LC > 0.7 (ref > 10) retention (ref > 10)

Table 1. Detailed Summary of Compounds Identified As Level 1 Structures

4 (1)

160 (2)

11 (1) 68 (5)

11(8)

11(8)

34 (26)

24 (12)

19 (8)

16 (15)

33 (3)

no. of candidates complying with both methods (ref > 10)

6,7-dimethoxy-2-(1piperazinyl)-4quinazolinamine telmisartan

2,3phenazinediamine 2,8phenazinediamine L-(+)-ergothioneine propranolol

tryptophan

guanine phenylalanine tyrosine

4- hydroxyquinoline

2-hydroxyquinoline

4-dimethyl aminopyridine 3-formylindole

compd name

1/1.0 (589)

1/0.73 (27) 1/1.00 (21024) 1/0.74 (73)

8/0.70 (11)

1/0.72 (611) 1/1.0 (13531) 1/0.97 (63897) 1/0.88 (22191) 1/0.70 (118)

2/0.83 (443)

1/0.87 (434)

3/0.95 (335)

1/0.96 (520)

rank/score (ref no.) in MetFrag Lists

antihypertensive drug

metabolite of doxazosine

natural antioxidant beta-blocker drug

dye precursor

dye precursor

amino acid

precursor for agrochemicals, pharmaceuticals precursor for antibacterial agents precursor for antibacterial agents nucleobase amino acid amino acid

catalyst

use/origin

Environmental Science & Technology Article


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Figure 3. Correlation of (a) 6,7-dimethoxy-2-(1-piperazinyl)-4-quinazolinamine concentrations and (b) total 2,3- and 2,8-phenazinediamine concentrations detected in BR extracts with the observed mutagenicity of the respective wastewater effluent extracts (given in rev./mL wastewater).

samples.11 The concentration of the doxazosine metabolite 6,7dimethoxy-2-(1-piperazinyl)-4-quinazolinamine (R2 = 0.85) and the summed concentrations of 2,3-and 2,8-phenazinediamine (R2 = 0.89) correlated linearly with the observed mutagenicity as shown in Figure. 3. All other compounds showed R2 values between 0.09 and 0.45. All three compounds were tested with TA98 strain applying external metabolic activation. 6,7-Dimethoxy-2-(1-piperazinyl)4-quinazolinamine did not show any activity in the tested concentration range (0.006−20 ng/μL medium). However, the parent compound doxazosine has been shown to induce genotoxic effects in the homologous recombination mechanism of DNA39 although it has been reported as inactive in the Ames test with strain TA98 and TA100 with and without metabolic activation.41 The correlation of the peak area of the doxazosine transformation product with mutagenicity together with literature findings suggests that the metabolite might contribute to the activity via a comutagenicity mechanism in the presence of other promutagens, which might be of relevance for aromatic amines as shown recently.35 However, this hypothesis was not tested in this study. Both 2,3- and 2,8-phenazinediamine showed strong mutagenicity in the Ames test with S9 in the concentration ranges detected in the BR extracts with 2,3-phenazinediamine being 4.5 times more mutagenic than 2,8-phenazinediamine (Figure S1), which is in compliance with the literature.42 The quantitative potencies of the compounds were evaluated with the slopes calculated from the fitted concentration−response curves (Table S2), and their contributions to the observed effects of the BR extracts are shown in Figure 4. The isomer 2,8-phenazinediamine contributed 86% and 28% to the observed mutagenicity of BR1 and BR2 extracts, respectively, while 2,3-phenazinediamine was not detected in these extracts. Both isomers together accounted for 47%, 4.9%, and 4.2% of the mutagenicity of BR4, BR5, and BR6, respectively. For BR3, the mutagenicity of both compounds at the concentrations detected corresponded to 400% of the observed mutagenicity. This extract was characterized by the highest load with organic compounds (least transparent extract) suggesting that the mutagenicity of BR3 extract might have been partially masked by cytotoxic or other inhibiting effects. Previous studies on 2,3- and 2,8-phenazindiamine focused mostly on their formation in commercial hair dyes containing ophenylenediamine (OPD) and p-phenylenediamine, respec-

fragments, and the molecular formulas of 72 respective analytes were assigned with high confidence. A fraction of 35 of the assigned formulas showed no aromatic structure in the Chemspider database search and was eliminated from further data evaluation. The 37 remaining analytes were assigned to 33 different molecular formulas (i.e., several isobaric compounds occurred). Compound Identification. For 23 out of 37 analytes, we obtained reference standards, and 14 compounds from different classes could be identified (Table 1). These compounds include four industrial chemicals, 4-dimethylaminopyridine, 3-formylindole, and 2,3- and 2,8-phenazinediamine; three amino acids, phenylalanine, tyrosine, and tryptophan, which are also used as nutritional supplements; two pharmaceutical precursors, 2- and 4-hydroxyquinoline; two pharmaceuticals, propranolol and telmisartan; the metabolite of the pharmaceutical doxazosine 6,7-dimethoxy-2-(1-piperazinyl)-4-quinazolinamine; the nucleobase guanine; and the natural antioxidant L-ergothioneine, which is frequently used in personal care products. Doxazosine, used to treat acute urinary retention in benign enlargement of prostate,39 was tentatively identified (level 2 according to Schymanski et al.38) by a comparison with the MassBank40 spectrum. Compounds were detected in all six extracts except for 2,3-phenazinediamine and 2-hydroxyquinoline which were detected in four extracts. Table 1 summarizes the stepwise reduction of candidates by the complementary LC methods and the details of the final level 1 identified compounds. The MS/MS spectra of these compounds and the respective reference standards are given in the SI, section S2.1. We tried to confirm nine analytes either with the highest ranked candidate fitting the experimental data or with one of the three highest ranked candidates that are known to be produced or were detected in the area, but none of them had a match with the obtained reference standards. Among the remaining 14 analytes, ten could not be confirmed since obtaining a reference standard would have required high efforts, although six out of these ten had only one candidate structure left complying with all preset criteria. Details of the unidentified compounds are summarized in Table S1 in the SI including information on the best matching structure if applicable. Unraveling the Mutagens and Their Contribution to the Observed Effects of the BR Extracts. Three candidate mutagens were identified by investigating the correlation of the concentrations of the identified compounds in the BR extracts with the previously reported mutagenic activities of the water 4685


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pounds, bioassay results, and unidentified compounds (PDF)

AUTHOR INFORMATION

Corresponding Author

*Phone: +49-341-235 1823. Fax: +49-341- 235-45-1530. E-mail [email protected]. ORCID

Melis Muz: 0000-0001-6510-5864 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was funded by the EDA-EMERGE project (FP7PEOPLE-2011-ITN, grant agreement 290100) and the SOLUTIONS project (grant agreement 603437), both supported by the EU Seventh Framework Programme. The authors thank Moritz Sievers for collecting and testing the BR extracts, Christine Hug for her valuable comments, and Margit Petre for her support with the Ames tests. Chemaxon (Budapest, Hungary) is acknowledged for providing an academic license of JChem for Excel, Marvin, and the Calculator Plugins.

Figure 4. Mutagenic activity of the BR extracts and diaminophenazine isomers detected in each BR extract expressed by the slope as revertants/mg BReq calculated by eq S1.

tively, when treated with hydrogen peroxide (H2O2) and found high mutagenicity with strain TA98 42,43 and the Oacetyltransferase overexpressing strain YG1024 with S9.44 2,3Phenazindiamine also showed significant genotoxic effects in an in vivo test with Drosophila melanogaster.45 Although OPD is banned for use in hair dye products, it is still a very important precursor for pesticides. Studies showed that 2,3-phenazinediamine is formed during the synthesis of the pesticides carbendazim and benomyl.46,47 Moreover, both 2,3- and 2,8phenazinediamine are precursors of diaminophenazine-based safranin and indulin dyes.48 These dyes were found to be produced in the industrial area of Bitterfeld, which could be a plausible source of these contaminants in the river. To the best of our knowledge, this is the first time that diaminophenazines are reported as environmental mutagens in surface waters. Although several studies suggest that aromatic amines are abundant contaminants in surface waters probably contributing to mutagenicity,49,50 no predominant mutagenic aromatic amine was associated with the observed effects. In this study, we could identify heterocyclic and aromatic amines from different compound classes in six BR extracts of river samples receiving discharges from a large industrialized area. The isomers 2,3- and 2,8-phenazinediamine were identified as novel environmental contaminants being highly mutagenic with the TA98 strain in the presence of S9 activation, and these compounds contributed strongly to the observed mutagenicity of the BR extracts and correlated with the mutagenicity of the solid phase extracts of grab water samples collected in parallel in a previous study. Considering the various sources and their mutagenic potencies, diaminophenazines should be carefully monitored in surface waters particularly those receiving wastewater effluents from dye industries. The presented results indicate a combination of effect-based tools,51 and targeted screening of specific compound groups is required for safe monitoring of hazardous substances in the environment. Specific sampling and extraction tools such as BR for aromatic amines together with diagnostic derivatization and candidate selection tools may be used to achieve the monitoring goals.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.est.7b00426. Detailed information on synthesis of 2,8-phenazinediamine, LC-HRMS analysis, spectra of identified com4686


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Identification of Mutagenic Aromatic Amines in River Samples with

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