Identification and quantitative confirmation of dinitropyrenes and 3-nitrobenzanthrone as major mutagens in contaminated sediments

Environment International 44 (2012) 31–39

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Identification and quantitative confirmation of dinitropyrenes and 3-nitrobenzanthrone as major mutagens in contaminated sediments Urte Lübcke-von Varel a, Mahmoud Bataineh a, Stefanie Lohrmann a, Ivonne Löffler a, Tobias Schulze a, Sini Flückiger-Isler b, Jiri Neca c, Miroslav Machala c, Werner Brack a,⁎ a b c

UFZ Helmholtz Centre for Environmental Research, Leipzig, Germany Xenometrix AG, Allschwil, Switzerland Veterinary Research Institute, Brno, Czech Republic

a r t i c l e

i n f o

Article history: Received 9 September 2011 Accepted 17 January 2012 Available online 13 February 2012 Keywords: Effect-directed analysis Mutagen identification Nitro-PAH Ames fluctuation assay LC–(APCI-)MS/MS

a b s t r a c t Polar fractions of a sediment extract of the industrial area of Bitterfeld, Germany, have been subjected for effect-directed identification of mutagens using the Ames fluctuation assay with TA98. Mutagenicity could be well recovered in several secondary and tertiary fractions. Dinitropyrenes and 3-nitrobenzanthrone could be confirmed to contribute great shares of the observed mutagenicity. In addition, a multitude of polar polycyclic aromatic compounds has been tentatively identified in mutagenic fractions including nitro-PAHs, azaarenes, ketones, quinones, hydroxy-compounds, lactones and carboxylic acids although their contribution to mutagenicity could not be quantified due to a lack of standards. Diagnostic Salmonella strains YG1024 and YG1041 were applied to confirm the contribution of nitro-aromatic compounds. We suggest the inclusion of dinitropyrenes and 3-nitrobenzanthrone into sediment monitoring in order to minimize the mutagenic risk to aquatic organisms and to human health. © 2012 Elsevier Ltd. All rights reserved.

1. Introduction Sediments can act as a reservoir for toxic compounds causing a range of adverse effects to biota. They are known to accumulate a broad range of substances like polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), naphthalenes (PCNs) and dibenzo-p-dioxins and -furans (PCDD/Fs). All of these compounds are nonpolar in nature and have been in the focus of sediment risk assessment for a long time (Brack et al., 2008; Fernandez et al., 1992). However, there is increasing evidence that more polar compounds, which have been largely neglected so far, may play an important role in sediment contamination and toxicity (Lübcke-von Varel et al., 2011; Terzic and Ahel, 2011). Effect-directed analysis (EDA) applying a stepwise fractionation procedure according to different physicochemical properties with subsequent biotesting and chemical analysis is a powerful tool to characterize and identify biologically active compounds (Brack, 2003). The reduction of the chemical complexity of fractions facilitates chemical analysis, reduces the number of potentially active compounds and minimizes antagonistic and synergistic effects and thus enables a reliable identification and confirmation of active components. Combining the EDA approach with the application of specific diagnostic strains indicating specific modes of action and thus groups of mutagens

⁎ Corresponding author. E-mail address: [email protected] (W. Brack). 0160-4120/$ – see front matter © 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.envint.2012.01.010

further supports the identification of causative compounds (Umbuzeiro et al., 2005). In a previous study organic sediment extracts from three contaminated sites of the river Elbe basin have been shown to elicit mutagenic, aryl hydrocarbon receptor (AhR)-mediated, tumor promoting and endocrine disrupting effects (Lübcke-von Varel et al., 2011). One of them, the Spittelwasser sample taken downstream of Bitterfeld (Germany) was further investigated in this study. Bitterfeld is one of the largest chemical industrial sites in Germany since more than 100 years and significantly contributes to the pollution of River Elbe with a multitude of chemicals (Brack and Schirmer, 2003; Brack et al., 1999; Stachel et al., 2005). The first characterization based on chromatographic separation according to a compound's polarity revealed that the majority of highly mutagenic fractions are medium-polar to polar whereas nonpolar compounds contributed only to a minor extent to the observed effects (Lübcke-von Varel et al., 2011). Chemical analysis detected more than 200 compounds and compound groups including potentially mutagenic, AhR-active, tumor promoting and endocrine disrupting substances such as PAHs and their nitrated and oxygenated derivatives, triclosan, 17β-estradiol and estrone. However, their actual individual contributions to the observed effects caused by the sediment extracts remained unidentified. Since the previous study indicated major mutagenic activity in the more polar primary fractions F14 and F15 of the Bitterfeld sediment extract (Lübcke-von Varel et al., 2011) the present study focuses on these primary fractions aiming to identify individual mutagenic compounds and to confirm their contribution to the mutagenic potency of

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the sediment. For this purpose, both primary fractions were subjected to a secondary fractionation step according to lipophilicity using reversed phase (RP), and F14 secondary fractions were further separated in a tertiary fractionation step using normal phase (NP) liquid chromatography on pyrenyl-bonded silica. All fractions were screened for their mutagenic potencies in the Ames fluctuation assay. In the previous study the most widely used strains TA98 and TA100 have been used, which are sensitive to compounds causing frameshift and base pair mutations, respectively (Lübcke-von Varel et al., 2011). Since strain TA98 indicated greater mutagenicity for all sediment extracts and fractions compared to TA100, the present study applied only diagnostic strains detecting frameshift mutations (TA98, YG1041 and YG1024). Typical mutagenic sediment contaminants include aromatic amines and nitro-compounds. For both of them acetylation of the N-hydroxylamine is a crucial metabolic step in the formation of the directly mutagenic agent (Colvin et al., 1998). The formation of N-hydroxylamine from nitroaromatic compounds is catalyzed by nitroreductase (Djuric et al., 1988). Thus, the strain YG1041 overproducing nitroreductase and Nhydroxylamine-O-acetyltransferase (OAT) (Hagiwara et al., 1993) as well as YG1024 only overproducing OAT (Watanabe et al., 1990) were used to characterize the contribution of aromatic amines and nitroaromatic compounds to mutagenicity. Mutagenic subfractions were analyzed for target analytes and unknowns using HPLC–MS/ MS and GC–MS/MS techniques. Detected mutagens were quantified and their mutagenic potencies were compared with those of the respective fraction to confirm their mutagenic input.

2. Materials and methods 2.1. Sample collection, sample preparation and primary fractionation The sediment sample was collected in a slowly flowing stretch of the river Spittelwasser using an Ekman-Birge-grab. A scheme of the sample preparation procedure is shown in Fig. 1. The sediment sample was freeze-dried, sieved (≤63 μm) extracted, cleaned up and primary fractionated applying an automated multi-step NP-fractionation method as described in detail elsewhere (Lübcke-von Varel et al., 2008, 2011). Two polar primary fractions F14 and F15 predominated mutagenicity and were selected for secondary fractionation. For capacity reasons, tertiary fractionation was restricted to the more complex F14 secondary fractions. Preliminary analysis suggested nitro-, ketoand hydroxy-PAHs and nitrogen-containing heterocyclic aromatic compounds as major components of these fractions (Lübcke-von Varel et al., 2011). 2.2. Secondary fractionation (RP-HPLC) For secondary fractionation F14 and F15 were dissolved in acetonitrile (ACN) and further separated by RP-HPLC using an HPLC system (ProStar 210, Varian Analytical Instruments, Darmstadt, Germany) equipped with a diode array detector operated from 250 nm to 400 nm and a fraction collector. Compounds were separated on a C18 stationary phase (Nucleosil 100-5C18 HD, 250×21 mm, Macherey-Nagel, Düren, Germany) using ACN and a buffer solution (0.05% CH3COONH4/acetic

Sieved sediment < 63 µm

Accelerated solvent extraction (ASE), solvents: DCM, acetone Accelerated membrane-assisted clean-up (AMAC)

Primary fractionation (NP separation on three connected columns) F1 to F12: nonpolar compounds (e.g. PCBs, PCDD/Fs, PAHs)

F13 to F18: compounds with increasing polarity (e.g. nitro-, oxy-, hydroxy-PAHs, N-heterocycles) F14, F15

Secondary fractionation (RP separation on C18) F14-1 to F14-17 F15-1 to F15-17

Tertiary fractionation (NP separation on pyrenyl column) F14-6-1 to F14-6-12 F14-7-1 to F14-7-12 F14-8-1 to F14-8-12 F14-9-1 to F14-9-12 F14-11-1 to F14-11-12 Fig. 1. Sample preparation procedure. NP: normal phase, RP: reversed phase, F: fraction, PCB: polychlorinated biphenyl, PCDD/F: polychlorinated dibenzo-p-dioxin/furan, PAH: polycyclic aromatic hydrocarbon.

U. Lübcke-von Varel et al. / Environment International 44 (2012) 31–39

acid, pH 4.75) as mobile phases at a flow rate of 10 ml min − 1. Gradients were optimized for both primary fractions with the more polar fraction F15 starting with 20% ACN followed by a slow gradient to 50% ACN within 90 min and a faster gradient within 20 min to 100% ACN. The less polar fraction F14 was separated with a gradient from 30% ACN to 60% ACN within 70 min and subsequently to 100% ACN within 20 min. In both programs the column was finally eluted with ACN for at least 30 min to avoid any memory effects. The fractionation windows were selected according to the occurrence of major peaks. Secondary fractions obtained from RP-HPLC were diluted with the buffer solution to an ACN/buffer ratio of 0.05 and re-extracted from the aqueous solution by solid phase extraction (SPE) using 60 mg of a mixed-mode solid phase consisting of polystyrene-divinylbenzene (Chromabond Easy, Macherey-Nagel, Düren, Germany) and 200 mg of an end-capped C18 stationary phase (Discovery DSC-18, Supelco, Taufkirchen, Germany). During extraction cartridges and sample vessels were covered with aluminum foil to avoid light induced decomposition of substances. After extraction of the first fraction (F1A, acidic fraction) of F14 and F15 the sample pH was adjusted to 10 with sodium hydroxide and extracted again (F1B, alkaline fraction). Cartridges were freeze-dried for at least 24 h and eluted with 5 ml HX, 10 ml DCM and 10 ml ACN, respectively.

33

TA98 counting the number of positive wells at a test concentration of 400 mg sediment equivalents (SEQ) ml− 1 (secondary fractions) and 800 mg SEQ ml− 1 (tertiary fractions), respectively. The significance of mutagenic effects was tested with the Wilcoxon–Mann–Whitney test with α = 0.5. For F14 and F15, mutagenic priority secondary and tertiary fractions, for reconstituted fractions and for individual candidate compounds concentration–response curves were determined applying concentrations from 1.2 mg SEQ ml − 1 to 800 mg SEQ ml− 1 and dilution factors of 2 and 3. Quantitative assessment of mutagenic potencies of fractions and confirmation of candidate compounds were based on slopes of the initial linear part of concentration–response curves and expressed as revertant wells per g sediment equivalent quantities (rev/g SEQs). Slopes and corresponding standard errors were derived from linear regression of all data pairs in the linear range with at least three replicates per concentration. All slopes were significantly different from 0 with a P b 0.001. Standard errors are given as error bars in Figs. 5 to 8. In comparison to the classical Ames test it should be considered that the test design allows for a maximum of 48 revertant wells (FlückigerIsler et al., 2004) in contrast to almost infinite numbers of revertants on the agar plate. Thus, one revertant well in the Ames fluctuation test may contain several revertant cells, which would be individually visible in the classical Ames test.

2.3. Tertiary fractionation (NP-HPLC) 2.5. Chemical analysis For tertiary fractionation selected RP fractions were dissolved in HX and separated on a pyrenyl-bonded silica stationary phase (250 × 10 mm, 5 μm Cosmosil PYE, average pore diameter 120 Å, Nacalai Tesque, Kyoto, Japan) in NP-mode using HX and DCM as mobile phases. A gradient elution program was applied starting with 100% HX held for 10 min, followed by a gradient to 15% DCM in 20 min and to 100% DCM in 30 min, which was held for another 30 min. Fractionation windows were chosen according to the occurrence of peaks. Reconstituted samples were prepared combining equal amounts of each associated fraction allowing the estimation of recoveries of mutagenic compounds. 2.4. Mutagenicity test — Ames fluctuation assay

50 ** * *

50 ** *

F14

40

*

40

* * **

30

2.5.2. LC–MS/MS Chromatographic separations were performed on an HPLC system equipped with a Surveyor MS pump and Surveyor autosampler (Thermo Electron, San Jose, CA, USA) and a polymeric RP column (Supelcosil LC-PAH, 250 × 2.1 mm, 5 μm, 120 Å, Supelco). A gradient elution program using water with 5% (v/v) methanol (A) and methanol (B) at a flow rate of 200 μl/min was applied starting with 40% B, held for 2 min and ramped to 100% B in 36 min, which was held for another 8 min and followed by equilibration. The column oven was set to 40 °C. Injection volume was 5 μl. Detection was performed on a linear trap quadrupole (LTQ) Fourier transform ion cyclotron resonance

F15 *

*

*

30

*

* 20

*

*

20

*

* 10

*

**

**

10

* *

* *

*

* *

* *

* * **

0 par rec 1A 1B 2 3 4 5 6 7/8 9 10 11 12 13 14 15 16 17

0

par rec 1A 1B 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

number of positive wells

The Ames fluctuation assay was performed with tester strain TA98 with and without metabolic activation as described before using 2aminoanthracene and 2-nitrofluorene as positive controls, respectively (Perez et al., 2003). Since experiments with TA98 revealed an activity reduction with S9 the strains YG1024 and YG1041 have been applied only without activation in the commercial Ames MPF™ fluctuation assay using 2-nitrofluorene and 1-nitropyrene as positive controls, respectively (Flückiger-Isler et al., 2004). Both test systems are very similar and have been tested to provide congruent results (data not shown). Concentrations are given as assay concentrations in the 24-well plates. Priority fractions for further investigation were identified with

2.5.1. GC–MS A gas chromatograph (GC, Agilent 6890, Agilent technologies, Santa Clara, CA, USA) coupled to a mass selective detector (MS, Agilent 5973) was equipped with an autosampler (Agilent Series 7683) and an HP5MS fused silica capillary column (35 m × 0.25 mm i.d., 0.25 μm, Agilent) with helium as carrier gas at a constant flow rate of 1.3 ml min − 1. Aliquots of 1 μl of sample were injected in splitless mode at 250 °C injector temperature. When used in scan mode, the oven temperature was as follows: 60 °C to 150 °C at 30 °C min − 1, then 6 °C min− 1 up to 186°, followed by 4 °C min− 1 to 280 °C and held for 21.5 min. Fractions were dissolved in toluene and analyzed using GC–MS in scan mode. Benzo[a]pyrene D12 was used as injection standard.

Fig. 2. Mutagenicity screening of secondary fractions with TA98 without (white) and with (black) S9 activation at a concentration of 400 mg SEQ ml− 1. par: parent fraction, rec: recombined mixture of all tertiary fractions. Fraction 1A: acidic, fraction 1B: basic fraction. Asterisks indicate a significant effect in the Wilcoxon–Mann–Whitney test.

number of positive wells

50 ** **

number of positive wells

U. Lübcke-von Varel et al. / Environment International 44 (2012) 31–39

50 **

number of positive wells

34

50

F14-6

50 * *

* *

40

40

30

30

20

20

10

* *

** *

*

* *

40

*

30

*

30

* **

0

** ** * **

20

*

* **

0

** *

*

10

par rec 1 2 3 4 5 6 7 8 9 10 11 12

F15-11

* ** *

*

30 * * * 20 *

*

30

* *

10

F15-12

50 * 40 *

* *

* *

20

* *

*

* *

10

* * *

0 par rec 1 2 3 4 5 6 7 8 9 10 11 12

par rec 1 2 3 4 5 6 7 8 9 10 11 12

0 par rec 1 2 3 4 5 6 7 8 9 10 11 12

*

20

40

**

*

0

50

*

par rec 1 2 3 4 5 6 7 8 9 10 11 12

par rec 1 2 3 4 5 6 7 8 9 10 11 12 10

par rec 1 2 3 4 5 6 7 8 9 10 11 12

F15-10

F15-9

*

0

40

0

30

20

*

* *

40

*

10

*

50 ** **

F14-11 *

30

* ** *

* *

10

40

20

* *

par rec 1 2 3 4 5 6 7 8 9 10 11 12

*

*

20

* **

50 ** *

F14-8

*

*

10

par rec 1 2 3 4 5 6 7 8 9 10 11 12

F14-9

40

10

50

*

0

0

30

F14-7

Fig. 3. Mutagenicity screening of tertiary fractions with TA98 without (white) and with (black) S9 activation at a concentration of 800 mg SEQ ml− 1. par: parent fraction, rec: recombined mixture of all tertiary fractions. Asterisks indicate a significant effect in the Wilcoxon–Mann–Whitney test.

(FTICR)-MS (Thermo Electron, Bremen, Germany) equipped with an atmospheric pressure chemical ionization (APCI) source as described in detail elsewhere (Bataineh et al., 2010). Secondary and tertiary fractions were diluted in MeOH to a final concentration of 33 g SEQ ml − 1 and 268 g SEQ ml − 1, respectively and analyzed for 55 target compounds representing nitro-PAHs, amino-PAHs, azaarenes, hydroxy-PAHs, keto-PAHs, quinones, and hydroxylquinones. These target compounds including mass spectrometric and chromatographic characteristics have been published previously (Bataineh et al., 2010). Quantification was based on fivepoint calibrations with concentrations ranging between 2 ng mL − 1 and 200 ng mL− 1. Each standard solution was injected three times.

F14-11, however with significant responses also in several other fractions (Fig. 4). The very similar responses of F14par, F14rec and F14add indicate excellent recovery of the compounds causing these effects and confirm the assumption of response additivity. Mutagenicity without external activation after tertiary fractionation of F14-6 to F14-11 was recovered in only two to four tertiary fractions (Fig. 3). Only these tertiary fractions have been subjected to quantitative mutagenicity assessment (Fig. 5). For F14-6 and F14-7 the activities of recombined mixtures significantly exceeded the responses to the parent fractions. Enhanced mutagenicity after fractionation, possibly indicating losses of mutagenicity masking or inhibiting compounds, is a frequently observed phenomenon (Brack et al., 2005; Iwado et al., 1991; Zeiger and Pagano, 1984). For F14-8 an excellent agreement between parent, recombined and summed responses was found, while for unknown reasons activities of F14-9rec and F14-11rec were significantly lower than those of the parent fractions. Additive responses of tertiary fractions F14-9add and

1000

3. Results and discussion 3.1. Mutagenicity assessment

rev/(g SEQ/ml)

800

-S9

600

400

200

F14-13

F14-12

F14-11

F14-10

F14-9

F14-8

F14-7

F14-6

F14-5

F14add

F14rec

0 F14par

In the first step, primary fractions F14 and F15, secondary fractions and recombined mixtures thereof, as well as tertiary fractions of most potent secondary fractions together with recombined mixtures have been screened for mutagenicity with TA98 in only one test concentration (400 mg SEQ ml− 1 and 800 mg SEQ ml− 1, respectively) in order to select potent fractions for further effect assessment and chemical analysis. Resulting preliminary mutagenicity patterns indicated only few secondary fractions (F14-6 to F14-9, F14-11, F15-9 to F15-13) contributing major mutagenicity to overall effects of the primary fractions (Fig. 2) and few tertiary fractions significantly contributing to overall effects of the secondary fractions (F14-6-9, F14-6-10, F14-7-8 to F14-7-10, F148-9 and F14-8-10, F14-9-9, F14-9-10 and F14-11-5 to F14-11-9) (Fig. 3). Subsequently, F14 and F15 as well as selected secondary and tertiary fractions were subjected to quantitative effect assessment applying concentration–response relationships and quantifying mutagenic potencies as slopes of the initial linear part of concentration–response curves as revertant wells/(g SEQ ml− 1). Assuming response additivity (Taylor et al., 1995), for primary and secondary fractions the responses of parent fractions (index par) were compared to the responses of recombined mixtures of all subfractions thereof (index rec) and to the mathematical sum of responses of mutagenic subfractions only (index add) as a recovery control of fractionation and to indicate possible antagonistic or synergistic effects. Quantitative assessment of F14 without external activation (−S9) confirmed greatest mutagenicity by F14-6 and

Fig. 4. Mutagenic response of F14 secondary fractions (gray), the parent fraction (par, black), the recombined mixture of all secondary fractions (rec, black) and the summed response of mutagenic fraction (add, black) without S9 activation in TA98.

U. Lübcke-von Varel et al. / Environment International 44 (2012) 31–39

500

160 140

400 -S9

120

300

rev/(g SEQ/ml)

rev/(g SEQ/ml)

35

200

100

+S9

100 80 60 40

F14-11par F14-11rec F14-11add F14-11-5 F14-11-6 F14-11-7 F14-11-8 F14-11-9

20

F14-11add similar to the responses of parent fractions suggest that low responses to recombined mixtures were not caused by losses during fractionation. Responses of F14 secondary fractions with external activation (+S9) indicate F146, F14-9 and F14-11 to contribute most to the response of F14 although mutagenicity was distributed over all secondary fractions indicating many different mutagens with different physico-chemical properties contributing to the overall effect (Fig. 6). The response of F14-6 with S9 (Fig. 3), could not be recovered in tertiary fractions and thus not used in the further analysis. The comparison of F14par with F14rec indicates a recovery of about 40% of mutagens requiring external activation. The slightly greater response to F14rec compared to F14add may be due to fractions F14-1 to F14-4 and F1414 to F14-17, which may contribute to the recombined mixture but were not included into quantitative mutagenicity assessment and thus the calculation of F14add due to low or nonsignificant responses in the screening. About 70% of the activity of F14-9par with S9 activation could be recovered in F149rec but only about 30% were contributed by tertiary fractions F14-9-9 and F14-9-10, which have been selected for quantitative mutagenicity assessment (Fig. 7). This suggests that other tertiary fractions contribute to mutagenicity. Also synergistic effects cannot be excluded. Similar to F14-9, mutagenicity of F14-11 with S9 activation was distributed over all tested tertiary fractions and thus, caused by many different mutagens with small individual contribution. A good agreement of F14-11par and F14-11add was found while F14-11rec exhibited about 200% response. Quantitative mutagenicity assessment of F15 secondary fractions indicated only a significant response without external activation (− S9, Fig. 8) with a recovery of about 60% in F15rec and good agreement of F15rec and F15add. Major mutagenicity was recovered in F15-9.

3.2. Compound identification in mutagenic tertiary fractions

F14-11-9

F14-11-8

F14-11-7

F14-11-6

F14-11-5

F14-11rec

F14-11add

F14-11par

F14-9-10

F14-9-9

F14-9rec

Fig. 5. Mutagenic response of F14 tertiary fractions (gray), the parent fractions (par, black), the recombined mixtures of all secondary fractions (rec, black) and the summed responses of mutagenic fraction (add, black) without S9 activation in TA98.

F14-9add

0 F14-9par

F14-9par F14-9rec F14-9add F14-9-9 F14-9-10

F14-8par F14-8rec F14-8add F14-8-9 F14-8-10

F14-7par F14-7rec F14-7add F14-7-8 F14-7-9 F14-7-10

F14-6par F14-6rec F14-6add F14-6-9 F14-6-10

0

Fig. 7. Mutagenic response of F14 tertiary fractions (gray), the parent fractions (par, black), the recombined mixtures of all secondary fractions (rec, black) and the summed responses of mutagenic fractions (add, black) with S9 activation in TA98.

mass and occurring fragments in MS2 experiments. For most peaks several isomeric structures possibly fitted to the mass spectrometric data. To provide realistic structure suggestions it was assumed that compounds that have been already found in the past and are listed in PubChem and other databases are more likely to occur in the investigated sediments. Thus, these databases were searched for the empirical formula resulting from exact mass spectrometry and cross checked for plausible fragments. For structure comparison PubChem CID numbers are given in Table 1. The majority of tentatively identified compounds are polycyclic aromatic ketones, quinones, hydroxy-compounds, lactones and carboxylic acids (Table 1). Although some of them have been shown to be mutagenic in different test systems (Durant et al., 1996), most of them do not appear to be significant mutagens in the Salmonella strains used in this study. Some of the compounds identified in this study, such as 7H-benz[de]anthracene-7one and 6H-cyclopenta[cd]pyrene-6-one, are well known weak mutagens in airborne particles (Durant et al., 1998) and sediments (Fernandez et al., 1992). However, for a lot of the tentatively identified compounds neither environmental nor toxicological data are available. Particularly due to the great share of quinones in the fractions some oxidative mutagenicity is expected (Chesis et al., 1984) although the applied strain TA98 is not very sensitive to this pathway. Because of the lack of mutagenicity data a quantitative estimate of the contribution of the tentatively oxygenated PAHs is not possible. Azaarenes and oxygenated derivatives thereof are a second class of compounds identified in mutagenic sediment fractions in this study. Among these compounds there are weak mutagens such as benzo[a]-, and benzo[c]acridine and dibenzo[a,j]acridine (Wood et al., 1983).

A broad variety of polycyclic aromatic compounds has been tentatively identified in mutagenic tertiary and (in the case of F15) secondary fractions based on the exact

700

2500 -S9

500

rev/(g SEQ/ml)

rev/(g SEQ/ml)

600

+S9

2000 1000

750

500

400 300 200

250

100

0

Fig. 6. Mutagenic response of F14 secondary fractions (gray), the parent fraction (par, black), the recombined mixture of all secondary fractions (rec, black) and the summed response of mutagenic fraction (add, black) with S9 activation in TA98.

F15-13

F15-12

F15-11

F15-10

F15-9

F15-8

F15add

F15rec

F15par

F14-13

F14-12

F14-11

F14-10

F14-9

F14-8

F14-7

F14-6

F14-5

F14add

F14rec

F14par

0

Fig. 8. Mutagenic response of F15 secondary fractions (gray), the parent fraction (par, black), the recombined mixture of all secondary fractions (rec, black) and the summed response of mutagenic fractions (add, black) without S9 activation in TA98.

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Table 1 Compounds tentatively (T) and positively (P) identified (iden) in tertiary fractions (frac) of F14 and secondary fraction of F15 using GC–MS (G) and LC–MS/MS (L). conc: concentration [ng/g SEQ], mut: mutagenic activity towards Salmonella strains with or without S9 activation, –: nonmutagenic, +: mutagenic, ?: inconclusive, *: activity towards structurally related isomers. Frac

Formula

Compound

CID

Conc Iden Mut

Ref

F14-6-9

C16H8N2O4

1,8-Dinitropyrene

39185

0.4

P/L

+

1-Hydroxypyrene Pyrenecarboxylic acid Cyclopenta[def]phenanthren-4-one carboxylic acid 5,12-Naphthacenequinone 1,6-Dinitropyrene

21387 125395

0.4

P/L T/L T/L T/L P/L

−

F14-6-10

C16H10O C17H10O2 C16H8O3 C18H10O2 C16H8N2O4

Rosenkranz and Mermelstein (1983) Lambert et al. (1995)

C16H10O C10H6O2 C10H8O C17H9O2 C16H7O3 C18H13O

1-Hydroxypyrene 1,4-Naphthoquinone Naphthol Pyrenecarboxylic acid Cyclopenta[def]phenanthrenone carboxylic acid Methylbenzo[a]fluoren-11-one isomer or methylbenzo[a]-phenalen-7-one 1-Hydroxypyrene 1-Acetylpyrene Dimethoxyanthracene Pyrene-1-cabonylcyanide 6-Benzoyl-2-naphthol 7-Methylbenzo[c]carbazole-1,4-dione Tetracene-5,6,11,12-tetrone 3-Methoxybenzo[a]phenalen-7-one 3,9-Dihydroxybenzo[a]phenalene-7-one 1,3-Dinitropyrene Cyclopenta[def]phenanthren-4-one Dimethylanthraquinone Acetylpyrene 1,6-Dinitropyrene

21387 8530 7005/8663 125395/334608

0.2

P/L T/G T/G T/L T/L T/L

− − −

Rosenkranz and Mermelstein (1983) Lambert et al. (1995) Sakai et al. (1985) Kier et al. (1986)

21387 96251 614085/83107/10944477 130232 93271 261065 303161

2.1

P/L T/G T/G T/L T/L T/L T/L T/L T/L P/L T/G T/G T/G P/L

−

Lambert et al. (1995)

+

Rosenkranz and Mermelstein (1983)

+

T/G T/L

+

Rosenkranz and Mermelstein (1983) Moller et al. (1985)

−

Salamone et al. (1979)

− +

Salamone et al. (1979) Chemical Safety Information (2009)

− +

Salamone et al. (1979) Chemical Safety Information (2009)

+ + −

Kier et al. (1986) Wislocki et al. (1976) Lambert et al. (1995)

F14-7-8

F14-7-9

F14-7-10

C16H10O C18H12O C18H18O2 C18H9ON C17H12O2 C17H11O2N C18H8O4 C18H12O2 C17H10O3 C16H8N2O4 C15H8O C16H12O2 C18H12O C16H8N2O4 C17H10O C19H14O3

F14-8-8

F14-8-9

F14-8-10 F14-9-9

F14-9-10 F14-11-5

Benzo[a]fluoren-11-one 2-Methyl-6-(naphthalene1-carbonyl)benzoic acid C21H11ON Dibenz[a,j]acridine-5,6-oxide C18H12O2 Benz[a]anthracene-7,12-dione C18H12O2 3-Methoxybenzo[a]phenalen-7-one C17H12O Naphthalen-1-yl(phenyl)methanone C19H10O2 Chryseno[4,5-bcd]pyranone C14H14O2N2 4,4′-Dimethoxyazobenzene C18H12O2 Benzo[a]anthracene-7,12-dione C17H11N Benzo[a]acridine C17H10O Benzo[a]fluorene-11-one C18H12O2 Benzo[a]anthracene-7,12-dione C17H11N Benz[a]acridine C19H10O 6H-Benzo[cd]pyrene-6-one and isomer C18H14O Terphenylol C20H12O Hydroxybenzo[a]pyrene, 4 isomers

C20H11O2 C20H10O2 C21H12O C20H12O C20H11NO2 C20H12O C16H10O C18H12O C22H14O C21H14O C20H17O C21H14O2 C22H16O

F14-11-6

C20H12O C19H14O C20H14O C20H14O2 C20H14O C19H10O2 C21H10O2 C16H9NO2 C16H8O2

Benzo[a]pyrene-1,3(2H,12aH)-dione Benzo[a]pyrene-6,12-dione Indeno[2,1-a]phenanthren-11-one Hydroxybenzo[a]pyrene, 2 isomers 6-Nitrobenzo[a]pyrene Hydroxybenzo[a]pyrene, 2 isomers 1-Hydroxypyrene 1-Hydroxychrysene 1-Benzo[e]pyren-4-ylethanone Benzo[a]pyren-6-ylmethanol Methylbenzo[b]phenanthren-12-yl methanol 6-Methyl-1,2-dihydrobenzo [j]aceanthrylene-9,10dione 10,14b-Dihydrobenzo[4,5]cyclo-hepta [1,2,3-de]anthracen-5(9H)-one 2H-Benzo[j]aceanthrylen-1-one phenyl-(4-phenylphenyl)methanone 12-Methylbenzo[b]phenanthrene-7-carbaldehyde (Benzoylphenyl)phenylmethanone 12-Methylbenzo[b]phenanthrene-7-carbaldehyde Chryseno[4,5-bcd]pyranone Perylo[1,12-b,c,d]pyranone 11H-Benzo[a]carbazole-1,4-dione Pyrene-1,8-dione

14160 39184

50605 53230 21963 317828/73698 96251/3015125/3013944/ 39184

1.5

0.3

0.1

10184

150126 17253 77267 69503 51320 10388 17253 9180 10184 17253 9180

0.7

0.6 0.6 0.5 0.1

350344 36636/42027/25890/32191/ 28598/37787/37880/42030/ 42029/42028 (a) 149474 18299 20729 (a) 44374 0.6 see above 21387 2.6 44285 188806 30544

T/L P/L T/L T/L T/L T/L P/L P/L T/L P/L P/L T/L T/G T/L

183235

T/L T/L T/L T/L P/L T/L P/L T/L T/L T/L T/L T/L

4145299

T/L

299463 75040 25894 70875/ 25894 51320 626296 261064 16820

T/L T/L T/L T/L T/L T/L T/L T/L T/L

+

U. Lübcke-von Varel et al. / Environment International 44 (2012) 31–39

37

Table 1 (continued) Frac

Formula

Compound

CID

Conc Iden Mut

Ref

+/ −

F14-11-7

F14-11-8

F14-11-9

F14-1110 F15-9

C20H11NO2

Nitrobenzo[a]pyrene

C20H12O C22H12O C22H12O2 C21H11N C20H13NO C21H12O2 C20H11NO2 C20H12O C22H12O C21H10O C21H11NO C20H11NO C20H14O C20H10O2 C21H11N C6H5ClN2O2 C15H8O C20H11NO2 C20H12O C22H10O2 C20H12O C20H12O2 C19H14O

Hydroxybenzo[a]pyrene Benzo[l]cyclopenta[cd]pyren-1(2h)-one Pentacene-6,13-dione Benzo[a]pyrene-6-carbonitrile 7H-Dibenzo[c,g]carbazol-2-ol 4H-Benzo[f]naphtho[2,1-c]chromen-4-one Nitrobenzo[a]pyrene, 2 isomers Hydroxybenzo[a]pyrene Indeno[1,2,3-cd]pyren-8-ol 11H-Cyclopenta[ghi]perylen-11-one Dibenz[a,j]acridine-5,6-oxide 9H-Benzo[f]indeno[2,1-c]quinolin-9-one 1-Benzo[c]phenanthren-5-ylethanone Benzo[a]pyrene-6,12-dione Benzo[a]pyrene-6-carbonitrile 2-Chloro-4-nitroaniline Cyclopenta[def]phenanthren-4-one Nitrobenzo[a]pyrene Hydroxybenzo[a]pyrene Anthanthrone Hydroxybenzo[a]pyrene Benzo[a]pyrene-7,8-diol 5-Hydroxy-7-methylbenz[a]anthracene

44374/50958/50961/ 154510 (b) (a) 188809 76415 186057 115202 376079 (b) (a) 150519 55051 150126 1210309 44275 18299 186057 8492 21963 (b) (a) 94183 (a) 42267 97402

C17H9NO3 C16H8N2O4

3-Nitrobenzanthrone 1,8-Dinitropyrene

2825690 39185

0.06 0.2

P/L P/L

+ +

C16H13N C17H11N C15H26O C17H10O C16H11N C14H8O2 C18H12O2 C18H10O C19H11N C18H11NO3 C18H10O C16H14O C17H11NO2 C19H12O C17H10O2 C19H12O2

N-Phenyl-2-naphthylamine Benzo[a]acridine Alpha-cadinol Benzo[a]fluorene-11-one Benzocarbazole Anthraquinone Benzo[a]anthracene-7,12-dione Cyclopenta[cd]pyrene-3[4H]-one Azabenzo[a]pyrene 7-Nitrobenz(a)anthracen-11-ol Benzo[ghi]fluoranthen-4-ol 1-(9,10-Dihydrophenanthren-2-yl)ethanone 7-Methylbenzo[c]carbazole-1,4-dione Benzo[a]anthracene-12-carbaldehyde 3-Hydroxybenzoanthrone 7-Methylbenzo[a]anthracene-3,4-dione

8679 9180 519662 10184 67459/9196/9202 6780 17253 149464 32463/9109/12435805 147335 10490347 220209 261065 145695 6697 150674

0.9 0.8

P/L P/L T/G T/G T/L P/L P/L P/L T/L T/L T/L T/L T/L T/L T/L T/L

− +

Enya et al. (1997) Rosenkranz and Mermelstein (1983) Chemical Safety Information (2009) Chemical Safety Information (2009)

−

Salamone et al. (1979) Salamone et al. (1979)

Highly potent nitro-PAHs such as dinitropyrene (DNP) isomers, 3-nitrobenzanthrone (3-NBA) and nitrobenzo[a]pyrenes have been detected in fractions with outstanding mutagenicity. 3-NBA and DNPs have been reported to be among the strongest known mutagens in the Ames test (Enya et al., 1997) and have been made responsible for large shares of mutagenicity in soil extracts (Watanabe et al., 1998). 3-NBA was detected in a concentration of 0.06 ng g− 1 SEQ in F15-9 while concentrations of 1,3-, 1,6- and 1,8DNP in the highly mutagenic fractions F14-6-9, F14-6-10, F14-7-9, F14-7-10 and F15-9 were in the 0.1 to 1.5 ng/g SEQ range. Thus, we hypothesized that 3-NBA and DNPs significantly contribute to mutagenicity of these fractions and subjected them to quantitative mutagenicity confirmation.

3.3. Quantitative mutagen confirmation For confirmation of individual toxicants as cause of mutagenicity of the fractions F14-6-9, F14-6-10, F14-7-9, F14-7-10 and F15-9 full concentration–response relationships were recorded for fractions and compounds. Quantitative confirmation was based on the initial linear slope of the curves (Table 2). The compounds 1,8-DNP and 1,6-DNP have been detected in fractions F14-6-9 and F14-6-10, respectively, in concentrations that cause twice the mutagenic response of the fractions when tested as individual compounds. Antagonistic (but also synergistic) effects in complex mixtures are a frequent observation (Hermann, 1981) that may confound quantitative confirmation in EDA. However, it is obvious that both DNP isomers may explain the majority if not all of the mutagenic effect of F14-6-9 and F14-9-10. In fractions F14-7-9 and F14-7-10 the compounds 1,3-DNP and 1,6-DNP explain 16% and 39% of the mutagenic response of the respective fractions. Thus, probably other nonidentified or non-quantified mutagens contribute to the mutagenicity of these fractions.

T/L T/L T/L T/L T/L T/L T/L T/L T/L T/L T/L T/L T/L T/L T/L T/L T/L T/L T/L T/L T/L T/L T/L T/L

In fraction F15-9 in addition to 1,8-DNP the extremely potent mutagen 3-NBA has been detected. Both compounds explain together more than 70% of the mutagenicity of this fraction (50% 1,8-DNP and 21% 3-NBA). Efforts were made to associate mutagenicity that could not be explained by the major mutagens 3-NBA and DNPs to other known or expected mutagens that could be quantified in the fractions. 6-Nitrobenzo[a]pyrene, 1-nitropyrene and 6-aminochrysene as well as the weakly mutagenic benzo[a]-, benzo[c]acridine and dibenzo[a,j]acridine were detected in the pg g− 1 SEQ and low ng g− 1 SEQ level in several mutagenic secondary and tertiary fractions of F14 and F15 (Table 1). The concentration of 6-nitrobenzo[a] pyrene, which was mutagenic after metabolic activation, was below the concentration causing effects in the assay in this study (49 ng ml− 1). Benzo[a]acridine and benzo [c]acridine did not show any mutagenic effect for the applied concentrations up to 20 μg ml − 1 and 0.4 μg ml− 1, respectively. 7-Nitrobenzo[a]anthracene and isomers of hydroxybenzo[a]pyrene, benzofluorenone, nitrobenzo[a]pyrene, 3-NBA and azabenzo[a] pyrene were detected in the highly mutagenic fractions F14-7-10, F14-11-8 and F15-9 and may have contributed to mutagenicity. 6- and 12-Hydroxybenzo[a]pyrene, 1- and 3-nitrobenzo[a]pyrene and 10-azabenzo[a]pyrene were reported to be mutagenic to tester strains TA98 and/or YG1041 (Chou et al., 1984; Wislocki et al., 1976; Yamada et al., 2002).

3.4. Mutagen confirmation via metabolization pathways In a second confirmation step tertiary fractions of F14 were tested without S9 activation with Salmonella strains YG1041 and YG1024. Both strains are derivatives of TA98 but designed to be more sensitive to nitroarenes and aromatic amines (Ohe et al., 2004). Nitro-aromatic compounds have been found to be activated by nitro group reduction and subsequent acetylation to form the ultimate mutagene, probably a

38

U. Lübcke-von Varel et al. / Environment International 44 (2012) 31–39

Table 2 Activities of mutagenic fractions and identified mutagens and their contribution to mutagenic effects detected in the Ames assay using TA98 without metabolic activation. 3-NBA: 3nitrobenzanthrone, DNP: dinitropyrene, C: concentration, rev: revertants, SEQ: sediment equivalents. Activity (A): slope of initial part of concentration–response curve. Fraction

Afraction [rev/(g SEQ ml− 1)]

Identified mutagens

Astandards [rev/(ng ml− 1)]

Cfraction [ng/g SEQ]

Astandards [rev/g SEQ ml–1]

Astandard/Afraction [%]

F14-6-9 F14-6-10 F14-7-9 F14-7-10 F15-9 F15-9

179 239 190 85 99 99

1,8-DNPa 1,6-DNPb 1,3-DNPa 1,6-DNPb 1,8-DNPa 3-NBAc

846 327 101 327 846 340

0.4 1.5 0.3 0.1 0.2 0.06

338 491 30 33 169 20

189 205 16 39 50 21

Tested concentration ranges. a 0.0004–400 ng ml− 1. b 0.016–16 ng ml− 1. c 0.006–12.5 ng ml− 1.

nitrenium ion (Aiub et al., 2006; Colvin et al., 1998). YG1041 overexpresses Oacetyltransferase and nitroreductase while YG1024 overexpresses only Oacetyltransferase (Umbuzeiro et al., 2004). A combination of these strains has been found to be useful to understand the role of nitro groups for mutagenicity (Umbuzeiro et al., 2005) and may also help to indicate the contribution of nitroaromatic compounds to mutagenicity of unknown mixtures. For 1,8-DNP Hagiwara et al. (1993) demonstrated a 20-fold greater response in YG1024 and an even 40-fold enhanced response in YG1041 compared to TA98. Since the Ames fluctuation assay allows only for a maximum response range between 1 and 48 compared to the much greater response rate of the classical Ames test similar response ratios cannot be expected. In our study highly mutagenic fractions F14-6-9, F14-6-10, F14-7-9, and F14-7-10, where DNP isomers were confirmed as causative compounds, exhibited 4- to 6-fold higher response with YG1024 (Fig. 9). Interestingly with YG1041 hardly any greater response was found for the DNP dominated tertiary fractions of F14-6 and F14-7. This is in contradiction to expectations from standard compounds and suggests that other compounds present in the fractions may suppress the mutagenic response. Overexpression of enzymes such as nitroreductase may also stimulate the generation of toxic products reducing growth and counteracting the mutagenic response. This phenomenon has been observed for example for N-nitroso compounds (Aiub et al., 2006) and may be an explanation for the findings in this study. The response of F14-11-6 increased 23fold with YG1041 suggesting nitro-benzo[a] pyrene or other non-identified nitro-compounds causing the effect. Responses of some fractions such as F14-7-8 and F14-11-8 did not show any increase in both strains. The greater response found with YG1024 confirms the role of nitro-PAHs for mutagenicity although the enhancement was less than expected. YG1041 completely failed to detect the DNPs in these fractions suggesting that false negative results due to interacting compounds in mixtures may confound the diagnosis.

4. Conclusions The compounds 1,3-, 1,6- and 1,8-DNP and 3-NBA were identified and quantitatively confirmed as predominating bacterial mutagens in this study. These compounds may play also a prominent role for human carcinogenicity since intestinal microorganisms may activate 25

Ratio

20

5

F14-11-9

F14-11-7

F14-11-8

F14-9-10

F14-11-6

F14-9-9

F14-8-9

F14-8-10

F14-7-9

F14-7-10

F14-7-8

F14-6-10

F14-6-9

0

Fig. 9. Response ratios of Salmonella strains YG1041/TA98 (black) and YG1024/TA98 (gray).

these compounds by their nitroreductases (Chadwick et al., 1992; Umbuzeiro et al., 2005). The importance of these mutagens is in agreement with previous studies (Enya et al., 1997; Watanabe et al., 1998) and suggests diesel exhaust and incomplete combustion, typically occurring at industrial sites, as the major cause of mutagenicity in Bitterfeld sediments rather than a site-specific production process. Thus, we may hypothesize similar problems at other industrial sites and suggest the inclusion of these compounds into future sediment monitoring to minimize mutagenic risks. In addition, a great number of other nitro-PAHs as well as hydroxy- and keto-PAH, polycyclic quinones and carboxylic acids, and azaarenes have been tentatively identified in mutagenic fractions. However, availability of standards for both analytical confirmation and evaluation of mutagenicity and toxicity is still very limited. For a better understanding of environmental mutagenicity and its impact on human health and ecological effects, such as reproductive consequences in aquatic organisms (Lacaze et al., 2011; Lewis and Galloway, 2009), it is crucial to unravel complex contamination with potentially mutagenic polar polycyclic aromatic compounds. Acknowledgments The presented work was supported by the European Commission through the Integrated Project MODELKEY (Contract-No. 511237GOCE). We thank Dr. Georg Reifferscheid for providing tester strain TA98. References Aiub CAF, Mazzei JL, Pinto LFR, Felzenszwalb I. Evaluation of nitroreductase and acetyltransferase participation in N-nitrosodiethylamine genotoxicity. Chem Biol Interact 2006;161:146–54. Bataineh M, Lübcke-von Varel U, Hayen H, Brack W. HPLC/APCI-FTICR-MS as a tool for identification of partial polar mutagenic compounds in effect-directed analysis. J Am Soc Mass Spectrom 2010;21:1016–27. Brack W. Effect-directed analysis: a promising tool for the identification of organic toxicants in complex mixtures. Anal Bioanal Chem 2003;377:397–407. Brack W, Schirmer K. Effect-directed identification of oxygen and sulphur heterocycles as major polycyclic aromatic cytochrome P4501A-inducers in a contaminated sediment. Environ Sci Technol 2003;37:3062–70. Brack W, Altenburger R, Ensenbach U, Möder M, Segner H, Schüürmann G. Bioassaydirected identification of organic toxicants in river sediment in the industrial region of Bitterfeld (Germany) — a contribution to hazard assessment. Arch Environ Contam Toxicol 1999;37:164–74. Brack W, Schirmer K, Erdinger L, Hollert H. Effect-directed analysis of mutagens and ethoxyresorufin-O-deethylase inducers in aquatic sediments. Environ Toxicol Chem 2005;24:2445–58. Brack W, Blaha L, Giesy JP, Grote M, Moeder M, Schrader S, et al. Polychlorinated naphthalenes and other dioxin-like compounds in Elbe River sediments. Environ Toxicol Chem 2008;27:519–28. Chadwick RW, George SE, Claxton LD. Role of the gastrointestinal mucosa and microflora in the bioctivation of dietary and environmental mutagens or carcinogens. Drug Metab Rev 1992;24:425–95. Chemical Safety Information from intergovernmental organisations. I.I.P.o.C.S. www. inchem.org2009. Chesis PL, Levin DE, Smith MT, Ernster L, Ames BN. Mutagenicity of quinones: pathways of metabolic activation and detoxification. Proc Natl Acad Sci 1984;81:1696–700.

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