Multidimensional monitoring of anaerobic/aerobic azo dye based wastewater treatments by hyphenated UPLC-ICP-MS/ESI-Q-TOF-MS techniques
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Sulfonated reactive azo dyes, such as Reactive Orange 107, are extensively used in textile industries. Conventional wastewater treatment systems are incapable of degrading and decolorizing reactive azo dyes completely from effluents, because of their stability and resistance to aerobic biodegradation. However, reactive azo dyes are degradable under anaerobic conditions by releasing toxic aromatic amines. To clarify reaction mechanisms and the present toxicity, the hydrolyzed Reactive Orange 107 was treated in anaerobic-aerobic two-step batch experiments. Sulfonated transformation products were identified employing coupled ICP-MS and Q-TOF-MS measurements. Suspected screening lists were generated using the EAWAG-BBD. The toxicity of the reactor content was determined utilizing online measurements of the inhibition of Vibrio fischeri. The OCHEM web platform for environmental modeling was instrumental in the estimations of the environmental impact of generated transformation products.
KeywordsICP-MS/ESI-Q-TOF coupling Online toxicity measurements Biological azo dye treatment Predicted reaction mechanisms and toxicity Transformation products Decolorization DOC removal
The remaining dyes from several industrial sources (e.g., textile, dye and dye intermediates, recycling, pharmaceuticals, etc.) are regarded as dischargers of a variety of organic pollutants into natural water resources or wastewater treatment systems (Carmen and Daniela 2012). Due to the high quantities of water used in the dying process, the textile industry is one of the biggest producers of liquid effluent pollutants (Saratale et al. 2011). Estimates show that 280,000 tons of textile dyes are discharged in industrial effluents per year worldwide (Jin et al. 2007). One group of these dyes are the sulfonated reactive azo dyes, which contain chromophoric azo groups, where nitrogen atoms are linked to sp2-hybridized carbon atoms of aromatic rings with additional sulfonic acid groups (Pathak et al. 2014). Plenty of treatment methods are applied to reduce the high amounts of colored wastewater. Chemical treatments (oxidation, electrolysis, ozonation) and physical treatments (filtration, adsorption, coagulation/flocculation) are generally applied, though biological treatments have the main advantage of converting over 70 % of organic matter (expressed by COD) to biosolids (Anjaneyulu et al. 2005). However, reactive textile dyes are not degradable in conventional aerobic treatment plants as they are persistent to biological oxidative degradation (Easton 1995). Yet, a degradation and decolorization of reactive azo dyes can be achieved under anaerobic conditions due to the cleavage of the azo bonds, with a further release of potential toxic and carcinogenic aromatic amines (Sweeney et al. 1994). With respect to the biological transformation products, several analytical LC-MS methods were reviewed for the analysis of sulfonated compounds (Reemtsma 2003) to clarify degradation/transformation mechanisms. Therefore, the coupling of ICP-MS and Q-TOF-MS for element and structure-specific identifications could be one further step in analyzing sulfonated transformation products. For monitoring the environmental impact of biologically treated substances, online toxicity measurements could be useful to improve long-term treatments in bioreactor systems. In addition, postulations of reaction mechanisms and interpretations of a present toxicity could be improved using several modeling databases.
Material and methods
Reactive azo dye: C.I. Reactive Orange 107
The industrial textile dye C.I. Reactive Orange 107 (CAS 90597–79-8) was kindly provided by DyStar (Leverkusen, Germany) for biological experiments. The hydrolyzed form of Reactive Orange 107 was prepared for 4 h at 80 °C after adjustment to pH 11 with 1 M L−1 sodium hydroxide and was neutralized with 1 M L−1 hydrochloride acid before treating.
The biological treatment was realized with an anaerobic-aerobic CSTR bioreactor system with a volume of 30 L each, according to previous studies of Plum and Rehorek (2005). Anaerobic and aerobic recirculating sludges were obtained from the industrial wastewater treatment plant of Currenta GmbH & Co. OHG (Leverkusen, Germany). The biomass was characterized by a dry matter of 25 g L−1 (DIN EN 12880 2001a), a sludge volume index of 20.6 mL g−1 (DIN EN 14702 2006) and by determining the loss on ignition of dry mass of 81.6 % (DIN EN 12879 2001b). The biomass was immobilized on polyurethane foamed carriers coated with activated carbon provided by LEVAPOR GmbH (Leverkusen, Germany) to improve biotransformation efficiency in both reactors, as reported in several studies (Yoo 2000; Pearce 2003; Srinivasan and Viraraghavan 2010; Vijayaraghavan et al. 2013; Kumar and Mongolla 2015). The bacterial consortium was conditioned for 10 days until immobilization rates >90 % were measured by determining the decreasing dry matter of the sludge according to DIN EN 12880 2001a. The pH was automatically adjusted to 7.0 ± 0.2 in both reactors. The temperature of the anaerobic treatment step was maintained at 38 ± 0.5 °C. Peristaltic pumps continuously recirculated the reactor content (100 L h−1) through ultrafiltration membrane cells in cross flow mode. The polyethylene membrane had a molecular weight cutoff of 50,000 Da, and the utilization and recovery rates for the observed compounds were tested in previous experiments by Plum and Rehorek (2005) and Rehorek et al. (2006). In addition to the online analytical set-up from Rehorek et al. (2006), an online UV-Vis spectrometer probe was installed with a flow cell in by-pass of the anaerobic bioreactor system for continuous decolorization measurements. Due to the involved cleaning procedure of the spectro::lyser probe (scan Messtechnik GmbH, Vienna, Austria), in combination with bacterial immobilization leading to a decreasing turbidity, spectrometric measurements could be carried out during the complete treatment time. The decolorization was calculated with the evaluation method (arithmetic mean from the spectral absorption coefficient of 436, 525, and 620 nm) of Döpkens and Krull (2004) according to the German wastewater regulation appendix 38. Furthermore, the actual DOC (dissolved organic carbon) could be measured online with the QuickTOC from LAR Process Analysers AG (Berlin, Germany).
In this study, 2 mM L−1 of the hydrolyzed Reactive Orange 107 was treated for 10 days under anaerobic conditions with a subsequent aerobic treatment of 7 days. The redox potential (against hydrogen potential) was continuously monitored during the anaerobic batch experiments. For Reactive Orange 107, a threshold of −330 mV for an anaerobic decolorization could be determined.
The determination of the transformation products of the hydrolyzed Reactive Orange 107 was performed employing the coupled UPLC-ICP-MS/ESI-Q-TOF-MS system.
Analytical set-up and measurement conditions for UPLC-ICP-MS/ESI-Q-TOF-MS analysis
Agilent 1290 Infinity
Agilent Poroshell 120 EC-C18 3 × 150 mm 2.7 μm
0.7 mL min−1
1 mM NH4Ac in H2O
5 mM NH4Ac in MeOH
99.5 % A
99.5–60 % A
60 % A
99.5 % A
Agilent Q-TOF 6530
11 L min−1
Sheath gas temperature
Sheath gas flow
10 L min−1
Agilent 8800 ICP-QQQ
10 % (80 % Ar/20 % O2)
Cell gas (O2)
0.35 mL min−1
m/z 32 (S+)
m/z 48 (SO+) product ion
Databases used for the prediction of biotransformation and environmental impact of biological azo dye wastewater treatments
The EAWAG-BBD (Biocatalysis/Biodegradation Database: Swiss Federal Institute of Aquatic Science and Technology, Duebendorf, CH; University of Minnesota) was used to create suspected lists for the spectroscopic screening of anaerobic and aerobic transformation products. The database contains information on microbial biocatalyzed and biodegradation pathways for primarily xenobiotic environmental pollutants (Ellis and Wackett 2012). With the primary focus on metabolic reactions, additional information on enzymes can be obtained via links to the Kyoto (Kanehisa et al. 2002), ExPASy (Gasteiger et al. 2003) and BRENDA (Scheer et al. 2011) databases. The pathway predictions are carried out by biotransformation rules, based on known chemical reactions of functional groups, like reduction, oxidation, elimination, or hydrolysis (Wicker et al. 2010).
The OCHEM web platform (online chemical modeling environment) was used to interpret the toxicity measurements during the biological treatment (Sushko et al. 2011). Besides biological activity and physicochemical properties of compounds, the database can predict information on toxicology and ecotoxicology effects for the bacteria Tetrahymena pyriformis, the most ciliated model used for laboratory research (Sauvant et al. 1999). In environmental toxicity prediction studies against T. pyriformis, a wide collection of 1093 experimental measurements were used for a critical assessment of QSAR (quantitative structure-activity relationship) models (Tetko et al. 2008).
Online toxicity measurements
During the textile dyeing process, hydrolysis (reaction that occurs in the presence of water) takes place in a competitive reaction to the dye fixation (Christie 2015). This leads to the hydrolyzed dye (RO107Hydrolyzate), that does not have any affinity with the fibers to form covalent bonds (Christie 2015), resulting in high concentrations in textile wastewater. Gottlieb et al. (2003) could show increasing toxicity for hydrolyzed dyes compared to the parent form with the bioluminescent bacterium Vibrio fischeri in a previous study. The decolorization of azo dyes under reductive conditions leads to aromatic amines, which can accumulate in the anaerobic treatment step (Brown and Laboureur 1983; Plum and Rehorek 2005; Phugare et al. 2011). In several studies, the increasing toxicity due to the release of aromatic compounds could be proven in separate offline toxicity measurements (Wang et al. 2002; Gottlieb et al. 2003; Işik and Sponza 2004; Sponza 2006; Işik and Sponza 2007; Solís et al. 2012). Therefore, the challenge was to develop an online toxicity measurement system, which can monitor the actual toxicity in the bioreactor during the complete treatment. The actual toxicity inside the reactor systems was measured with the Microtox® CTM (continuous toxicity monitor) from Modern Water (Cambridge, UK) with the bioluminescent bacterium V. fischeri. The common use of the instrument, according to the manufacturer, is the monitoring of rivers, lakes, reservoirs, seawater, recycled water, and groundwater (Modern Water, 2012). Toxic effects were determined by the inhibition of the bioluminescence of V. fischeri, measured with detectors inside the instrument after a contact time of 2 min. Offline samples of dyes and their transformation products were analyzed with the CTM and compared to common respiration tests with activated sludge. Although the results from respiration tests are more significant to evaluate real toxicity towards activated sludge bacterial community, evidenced correlations of measured and compared inhibitory effects provide the use of this online monitoring system. In addition, Gutiérrez et al. (2002) could show a higher sensitivity for bioluminescence inhibition in a comparative study between an offline Microtox® and respiration tests for seven organic toxic compounds.
To compensate the decreasing light output during the whole treatment/measurement process according to Michaelis-Menten kinetics for V. fischeri, reference values with distilled water had to be created for online measurements. Therefore, an electropneumatic valve was integrated to receive reference values (L0) while measuring. The cycle time for sample (Lt) and distilled water (L0) was set to a ratio of 45 and 15 min measuring time. Fifteen minutes was determined as a minimum switching time to receive constant inhibition values tested with 3.5-dichlorophenol from Sigma-Aldrich Chemie GmbH (Munich, Germany). An internally developed software was used to calculate the actual inhibition in real-time to monitor the toxicity during the treatment process. With this measurement set-up, online toxicity could be continuously measured for 4 weeks according to the decreasing bioluminescence of V. fischeri.
Results and discussion
Compound analysis by UPLC-ICP-MS/ESI-Q-TOF-MS techniques
Exact masses and mass shifts of the found compounds of the biologically treated samples with ion formula and abbreviations
Compounds of the hydrolyzed dye
Transformation products after anaerobic treatment
Transformation products after subsequent aerobic treatment
Degradation/transformation mechanisms of hydrolyzed Reactive Orange 107
Reactive Orange 107, as an aromatic compound with one –N=N– group, constitutes one representative of the largest class of synthetic dyes in commercial applications (Zollinger 2003). As an anionic monoazo reactive dye, Reactive Orange 107 has a sulfonate group (–SO3H) as an auxochrome group, which is ionizable and confers the binding capacity onto the textile material for the dye (Welham 2000).
In the anaerobic treatment step the cleavage of the azo bond could be observed for RO107Hydrolyzate and RO107Hydrolyzate-Ac through a decolorized sample. Under reductive conditions, 2-[(4-Aminophenyl)sulfonyl]ethanol (TFP2) was determined as a transformation product of both present dye forms. In addition, 2,4,5-triaminobenzenesulfonic acid (TFP3) was identified as a common transformation product of both dye forms. For the hydrolyzed RO107, 4-acetamido-2,5-diaminobenzenesulfonic acid (TFP1) could be determined as a primary breakdown product, which leads to TFP3 by hydrolysis of the acyl group. The eliminated group should be present as acetate due to the pH of the bioreactor system. All postulated transformation products could be observed with the LC-MS system (Fig. 2b).
In the subsequent aerobic treatment step, the anaerobic accumulated TFP2 was metabolized to the catechol 4-((2-hydroxyethyl)sulfonyl)benzene-1,2-diol (TFP2.1) by dioxygenase with loss of ammonia. Under aerobic conditions, the amine could be substituted completely, as described in previous studies (Viliesid and Lilly 1992; Blümel et al. 1998; Deniz and Grady 2003; Wang et al. 2006), so that TFP2 could not be found at the end of the aerobic treatment step (Fig. 2c).
For TFP1, a further elimination of the acyl group was observed under aerobic conditions. By acting on carbon-nitrogen bonds, amidohydrolase could lead to 2,4,5-triaminobenzenesulfonic acid (TFP3) under aerobic conditions (Hart and Orr 1975). In two subsequent steps, the transformation of TFP3 could be postulated. In several stages, the auxochrome amino groups was substituted by monooxygenase (Balba et al. 1979; Storm 2002; Stüber 2005), which leads to the aerobic accumulating 2,4,5-trihydroxybenzenesulfonic acid (TFP3.2), that was determined after the aerobic treatment.
In a parallel occurring transformation of TFP1 due to dioxygenase and monooxygenase, TFP1.1 and TFP1.2, which could not be determined, could be generated under loss of ammonia from the amine groups. Therefore, N-(2,4,5-trihydroxyphenyl)acetamide (TFP1.3) was found as an aerobic accumulating transformation product. The desulfonation of the aromatic sulfonate (TFP1.2) leading to the corresponding phenol (TFP1.3) could occur under sulfate limiting conditions, where aromatic sulfonates can be used as a primary s-source (Luther and Soeder 1987; Zürrer et al. 1987; Wittich et al. 1988; Kertesz et al. 1994; Ruff et al. 1999; Storm 2002). Compared to previous studies (Gottlieb et al. 2003; Supaka et al. 2004; dos Santos et al. 2007; Saratale et al. 2011; Balapure et al. 2014), a complete subsequent ring fission could not be observed in this experiment, although the treatment time of 7 days was much higher compared to conventional industrial wastewater treatment plants or lab-scale experiments (Işik and Sponza 2004; Spagni et al. 2010; Jonstrup et al. 2011; Muda et al. 2011).
Results of online toxicity measurements by V. fischeri during the treatment process
DOC, predicted toxicity, and relative concentration of the hydrolyzed dye forms and the biological transformation products
Pred. toxicity [log(IGC50−1)/mg L−1]
Relative concentration [%]
Rel. pred. toxicity [%]
DOC [mg L−1]
128.0 ± 1.50
17.1 ± 1.40
52.3 ± 1.40
Anaerobic transformation products
341.0 ± 1.30
575.0 ± 0.82
255.0 ± 1.30
Aerobic transformation products
298.0 ± 0.90
48.7 ± 0.93
255.0 ± 1.30
15.6 ± 1.10
The coupled ICP-MS and ESI-Q-TOF-MS systems were used to advance screening methods for the identification of sulfonated transformation products. With the help of the Q-TOF-data, the proposed molecules from the EAWAG-BBD were confirmed employing exact mass measurements. Ninety-seven percent decolorization was achieved under anaerobic conditions cleaving the present azo bonds. With the release of aromatic amines, an increasing toxicity compared to the initial dye product was determined with online measurements. Subsequently, decreasing toxicity in the aerobic treatment step was detected and could be explained by the fade of aromatic amines. Predicted toxicity values for the biological transformation products using the OCHEM web platform correlated with the inhibition values obtained employing online measurements. These results indicate that biological treatment of dyestuff can only be effective with the combination of anaerobic and aerobic treatment steps mediating efficient decolorization and detoxification.
We would like to thank the UFZ (Leipzig, Germany) for excellent support on ICP-MS and Q-TOF-MS measurements. Furthermore, we want to acknowledge Karl Mocha (University of Applied Sciences, Cologne) for the expert technical support in automated toxicity measurements. We are grateful to Modern Water for providing the Microtox® CTM utilized in online toxicity measurements.
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