Chemosphere 131 (2015) 34–40

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An efficient laboratory workflow for environmental risk assessment of organic chemicals Linyan Zhu a,b, Beatrix Santiago-Schübel a, Hongxia Xiao b, Björn Thiele c, Zhiliang Zhu d, Yanling Qiu d, Henner Hollert b,e,f,g, Stephan Küppers a,⇑ a

Research Center Jülich, ZEA-3, Jülich 52425, Germany RWTH-Aachen University, Aachen Biology and Biotechnology – ABBt, Institute for Environmental Research, Department of Ecosystem Analysis, Aachen 52074, Germany Research Center Jülich, Plant Sciences, Jülich 52425, Germany d Tongji University, Environmental Science and Technology Department, Siping Road 1239, Shanghai 200092, People’s Republic of China e State Key Laboratory of Pollution Control & Resource Reuse, School of the Environment, Nanjing University, Xianlin Avenue 163, Nanjing 210046, People’s Republic of China f College of Resources and Environmental Science, Chongqing University, Tiansheng Road Beibei 1, Chongqing 400030, People’s Republic of China g Key Laboratory of Yangtze Water Environment, Ministry of Education, Tongji University, Siping Road 1239, Shanghai 200092, People’s Republic of China b c

h i g h l i g h t s  A lab workflow for environmental risk assessment of chemicals and redox TPs was set up.  We validated the workflow with a sample substance-carbamazepine.  Oxidative transformation process of CBZ was simulated and fully studied by EC–MS.  The combined toxicity of CBZ and TPs was much higher than CBZ in fish embryo test.

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Article history: Received 19 December 2014 Received in revised form 12 February 2015 Accepted 14 February 2015 Available online 9 March 2015 Handling Editor: Caroline Gaus Keywords: Risk assessment Electrochemistry–mass spectrometry Ecotoxicity Organic pollutants Carbamazepine

a b s t r a c t In this study, we demonstrate a fast and efficient workflow to investigate the transformation mechanism of organic chemicals and evaluate the toxicity of their transformation products (TPs) in laboratory scale. The transformation process of organic chemicals was first simulated by electrochemistry coupled online to mass spectrometry (EC–MS). The simulated reactions were scaled up in a batch EC reactor to receive larger amounts of a reaction mixture. The mixture sample was purified and concentrated by solid phase extraction (SPE) for the further ecotoxicological testing. The combined toxicity of the reaction mixture was evaluated in fish egg test (FET) (Danio rerio) compared to the parent compound. The workflow was verified with carbamazepine (CBZ). By using EC–MS seven primary TPs of CBZ were identified; the degradation mechanism was elucidated and confirmed by comparison to literature. The reaction mixture and one primary product (acridine) showed higher ecotoxicity in fish egg assay with 96 h EC50 values of 1.6 and 1.0 mg L1 than CBZ with the value of 60.8 mg L1. The results highlight the importance of transformation mechanism study and toxicological effect evaluation for organic chemicals brought into the environment since transformation of them may increase the toxicity. The developed process contributes a fast and efficient laboratory method for the risk assessment of organic chemicals and their TPs. Ó 2015 Elsevier Ltd. All rights reserved.

1. Introduction Thousands of organic chemicals such as pesticides, pharmaceuticals, biocides and industrial chemicals are widely present in various environmental matrices due to their ubiquitous and intensive ⇑ Corresponding author. Tel.: +49 2461612766; fax: +49 2461612560. E-mail address: [email protected] (S. Küppers). http://dx.doi.org/10.1016/j.chemosphere.2015.02.031 0045-6535/Ó 2015 Elsevier Ltd. All rights reserved.

use (Biselli et al., 2005; Emelogu et al., 2013), and each year the number of new chemicals brought into the environment is increasing. Most of them undergo a series of chemical and/or microbial transformations which form a wide range of intermediates and transformation products (TPs). The TPs may have a different level of ecotoxicity compared with the parent compound, with so far widely unknown consequences on the environment and human health (Boxall, 2004). The presence of these organic chemicals

L. Zhu et al. / Chemosphere 131 (2015) 34–40

and their TPs in the environment is recognized to be one of the emerging environmental problems. Hence, it is of great importance to assess the potential environment risks of organic chemicals as early as possible. For risk assessment, it is indispensable to have a comprehensive understanding of transformation behavior of organic chemicals; especially the formation and environmental presence of various TPs and further complexity to risk assessment. Following the investigation of the transformation mechanisms, toxicity evaluation of the parent compounds and their TPs should be conducted to provide information on whether the toxicity is increasing or decreasing during the transformation processes, and hence possible toxic TPs. Although numerous researches have been carried out to investigate the transformations of the organic chemicals (Semple et al., 2001; Perelo, 2010), for many new substances, only limited information concerning their transformation behavior and TPs is available. This situation makes toxicity evaluation more challenging for risk assessment. In the present work, we established a laboratory workflow as a rapid screening tool for environmental risk assessment of organic chemicals undergoing redox transformation processes by integrating four processes: (i) Electrochemical simulation and redox mechanism studies of organic chemicals; (ii) Scaling up EC reactions; (iii) Purification and concentration of the reaction mixture; (iv) Evaluation of the toxicity of the parent compound and reaction mixture. It is known that the most important environmental transformation pathways of organic chemicals always involve a redox mechanism, which can be simulated electrochemically. In our previous works (Hoffmann et al., 2011; Chen et al., 2012, 2013), we have developed electrochemistry coupled online to mass spectrometry (EC–MS) as a fast and simple tool to simulate and investigate transformations of organic chemicals for both oxidation and reduction conditions in the environment. However, the small reaction capacity of EC–MS limits its usage for more environment issues. To break through the limit of EC–MS, a novel batch reactor – ‘‘synthesis cell’’ was introduced in the workflow to scale up the EC reactions which can obtain milligram amounts of reaction mixture. The reaction mixture containing high concentration buffer compound, formic acid and methanol cannot be directly used for toxicity tests. Therefore, a sample treatment process was also developed in this work to purify and concentrate the reaction mixture. Introducing the concept of ‘‘effect-driven approach’’ (Escher and Fenner, 2011), the combined toxicity of reaction mixture was evaluated for a fast screening of toxicity change during transformation processes, complemented with the toxicity information of parent compound and commercial TPs. In this case, the toxicity of TPs that are not structurally known and/or not commercial available can be assessed. Fish embryo test (FET) was applied for toxicity evaluation in this workflow, since FET has been found as sensitive as regular acute toxicity studies with adult fish (Lammer et al., 2009) and effectively used to evaluate the toxicity after ozonation (Magdeburg et al., 2014). With this workflow, it is possible to rapidly and simply assess new organic pollutants in laboratory, of which transformation mechanism and toxicity of TPs are unknown. To demonstrate the effectiveness of the workflow, carbamazepine (CBZ) was selected as a sample compound for the following reasons. First, CBZ was a widely used pharmaceutical and frequently detected in treated wastewater effluent and drinking water. Secondly, the transformation mechanism of CBZ has been extensively studied (Li et al., 2013; Mohapatra et al., 2014) which might facilitate TPs identification and comparison between reactions in the EC cell and in real situations. Finally, some of its TPs are known to be more toxic than the parent compound (Donner et al., 2013) which also facilitates a comparison with the toxicity results.

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2. Materials and methods 2.1. Chemicals and regents All solvents (chromatographic grade) and chemicals (analytical grade) were used as received from the commercial suppliers. Carbamazepine (98%, CAS 298-46-4) was purchased from Alfa Aesar and acridine (97%, CAS 260-94-6) was from Sigma–Aldrich. Ammonium acetate (p.a.), methanol (LiChrosolv purity) and dimethyl sulfoxide (DMSO) were purchased from Merck KGaA (Darmstadt, Germany). Formic acid was obtained from ROMIL (Cambridge, UK). Water of high purity (18.2 MX cm) was produced by a MilliQ plus 185 (Millipore, Molsheim, France). 2.2. EC–MS setup 2.2.1. Electrochemical reactions A commercial EC cell (Antec Leyden, The Netherlands) with a built-in platinum counter electrode and Roxy potentiostat was set up as reported in previous investigations (Hoffmann et al., 2011; Chen et al., 2012, 2013). The electrochemical reactions were conducted in a flowthrough ‘‘reactor cell’’ containing a working electrode (glass carbon, boron-doped diamond, Ag and Pt) and a pH-dependent HyREF electrode as a reference electrode. In the case of CBZ, boron-doped diamond (BDD) electrode was chosen as the working electrode for oxidation. The reaction solution was composed of 50 lM CBZ with a buffer of 10 mM ammonium acetate, in a mixture of methanol and water (3:2) containing 0.1% formic acid. All other conditions and experimental setup of EC–MS were as reported previously (Hoffmann et al., 2011; Chen et al., 2012). All reactions were repeated in triplicate to ensure that the system was stable and minimize bias and random errors. 2.2.2. Mass spectrometry conditions MS experiments were carried out on Q-TRAP 2000 ESI-MS/MS (ABSciex, Darmstadt, Germany) and high-resolution ESI-FTICR-MS Ultra (Fourier Transform Ion Cyclotron Resonance mass spectrometry) (ThermoFisher Scientific, San Jose, CA, USA), respectively. All conditions were used as reported previously (Hoffmann et al., 2011; Chen et al., 2012). 2.3. Preparation of reaction mixture for toxicity testing Scaling up reactions were conducted in a commercial available ‘‘synthesis-cell’’ (Antec Leyden, The Netherlands) consisting of three electrodes: the working electrode (WE), reference electrode (RE) and auxiliary electrode (AUX) with up to 80 mL of sample solution in a glass reaction vessel. The working electrode in our study is a flat smooth two-side BDD electrode (4.5  4.5  0.2 cm). 50 mL sample solution containing 1 mM CBZ, and 10 mM ammonium acetate with 0.1% formic acid, was degraded in the ‘‘synthesis cell’’ for about 2 h. The reaction solution was stirred throughout the reaction period by magnetic stirrer. 250 mL sample solution was collected by repeating the process five times. The solvent of the reaction mixture was removed by the SpeedVac system (Thermo Fisher Scientific, USA). Then the sample was dissolved in water and the pH of the sample solution was adjusted to 2. Solid phase extraction (SPE) was subsequently used to purify and concentrate the reaction mixture sample. Based on the structures and properties of CBZ and its TPs, C18 Hydra column (3000 mg, 6 mL, Macherey and Nagel, Düren, Germany) was selected as the solid phase. Each SPE column was conditioned with 80 mL MeOH and then 80 mL H2O (pH = 2). The sample solution was loaded on the columns at a flow rate 4–5 mL min1. Each column was then

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L. Zhu et al. / Chemosphere 131 (2015) 34–40

Fig. 1. Sample treatment process for the reaction mixture.

washed with 80 mL water, and the components were eluted with 60 mL MeOH containing 0.1% formic acid. Finally, the elution solution was dried using SpeedVac and the sample reconstituted in DMSO for toxicity tests. The workflow is shown in Fig. 1. The final sample was quantified in an Agilent1100 HPLC system interfaced with a Q-TRAP 4000 (ABSciex, Darmstadt, Germany). A C18 Eclipse Plus column (100  4.6 mm, 2.6 lm particle size, Agilent Technologies) was used for separation. Column temperature was set at 30 °C. The binary mobile phase consisted of (A) H2O with 0.1% formic acid and (B) acetonitrile with 0.1% formic acid. The 20 min gradient was as follows: 0–2 min 95% A isocratic, 2–12 min 5–95% B linear, 12–16 min 95% B isocratic, 16–20 min 95% A isocratic at a flow rate of 800 lL min1. The components were analyzed in the multiple reaction monitoring (MRM) mode. 2.4. Fish embryo test (Danio rerio) 2.4.1. Test design Maintenance of the zebrafish and egg production can be found in Hollert et al. (2003), Peddinghaus et al. (2012) and Lammer et al. (2009). The assay was carried out according to DIN 38415-6 (2009) and the methods detailed in Heger et al. (2012). Artificial water (ISO 7346-3, 1996) was used as exposure medium. All samples were prepared in DMSO and then diluted into different concentrations in exposure medium. The DMSO concentration in exposure medium was no more than 0.5%, which has been proven to have little or no adverse effects on the development of zebrafish embryos (Hallare et al., 2006). The positive control was 3,4-dichloroaniline (DCA; 3.7 mg L1, >98%; Sigma–Aldrich, Steinheim, Germany) dissolved in Millipore water. Three types of control were used: (1) negative control (artificial water), (2) DMSO control (artificial water with 0.5% DMSO), and (3) process control (prepared following the same process without adding the target compound). The embryos were exposed to the controls and different concentrations (Table S1) of CBZ, reaction mixture and acridine in 24-well plates, respectively. Each Egg was incubated individually in 2 mL exposure medium. Each chemical/sample was tested in at least three independent replicates with ten embryos per test concentration and controls. The temperature during exposure was kept at 26 ± 1 °C. The status of embryos was observed at 24, 48, 72 and 96 h, respectively. Mortality criteria were coagulation of embryos, a lack of heart function or a non-detachment of the tail. The lethal and sublethal effect endpoints were recorded as follows: coagulation, blastula, epiboly, somites, eye anlage, tail detachment, spontaneous movement of tail, heartbeat, blood circulation, pigmentation, teratogenic effects and edema. A test was considered valid if the mortality of the negative control (artificial water) did not exceed 10% and the positive control induced effects in more than 20% of the embryos. Samples that induced higher mortality than

10% were embryotoxic (DIN 38415-6, 2009). There was no significant difference discovered between process control and negative control. 2.4.2. Statistical analysis Data analysis of FET Test was conducted following the recommendations of Peddinghaus et al. (2012). Mortality/effect rate from triplicates were plotted using the software (Prism 6.0, GraphPad, La Jolla, USA). The half lethal concentration (LC50) and half maximal effective concentration (EC50) were computed from the concentration–response curves using log (agonist) vs. response with variable slope, where the top and bottom of the curve was respectively set to 0% (minimum of negative control) and 100% (maximum of positive control) and the significance was p < 0.05. 3. Results and discussion 3.1. Simulation of the transformation of CBZ by EC–MS 3.1.1. Electrochemical oxidation of CBZ The mass voltammogram of CBZ (m/z = 237) was recorded while a potential ramp from 0 to 3000 mV was applied with a slope of 10 mV s1, as shown in Fig. S1. The results indicated that the degradation of CBZ started in the EC cell at 1200 mV. To obtain more information of the TPs, the mass spectra of CBZ reacted at different voltages were measured. The mass spectra of the samples when EC voltages were 0 and 2000 mV were shown in Fig. S2. New peaks for possible TPs with masses [M + H]+ 180, 208, 210, 224, 226, 251 and 253 Da were observed. Based on the mass information of TPs, the mass voltammogram of these product ions (Fig. 2) were also measured for a potential ramp from 0 to 3000 mV to investigate the transformation trend of the TPs. When the voltage increased to 1200 mV, all the possible products started to form. The TPs with masses 180 Da and 208 Da first increased significantly and gradually reduced while the voltages kept increasing, which were converted into other TPs or mineralized when the voltage was higher than 1800 mV. The mass intensity of other TPs gently rose with the increase of EC voltage. Based on the information displayed in Fig. S2 and Fig. 2, 2000 mV was chosen as working voltage for the degradation experiments. 3.1.2. Mechanism study The structural information of the TPs was identified by EC coupled to high resolution FTICR-MS. The elemental composition/molecular formulas were created on the basis of the exact masses (error

An efficient laboratory workflow for environmental risk assessment of organic chemicals.

In this study, we demonstrate a fast and efficient workflow to investigate the transformation mechanism of organic chemicals and evaluate the toxicity...
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