Chemosphere 186 (2017) 652e659
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The impact of chemosensitisation on bioaccumulation and sediment toxicity Denise Kurth a, b, Stefan Lips a, Riccardo Massei a, b, Martin Krauss a, Till Luckenbach c, Tobias Schulze a, Werner Brack a, b, * a b c
UFZ d Helmholtz Centre for Environmental Research, Department of EffecteDirected Analysis, Permoserstr. 15, 04318 Leipzig, Germany RWTH Aachen University, Department of Ecosystem Analyses, Institute for Environmental Research, Worringerweg 1, 52074, Aachen, Germany UFZ d Helmholtz Centre for Environmental Research, Department of Bioanalytical Ecotoxicology, Permoserstr. 15, 04318 Leipzig, Germany
h i g h l i g h t s Zebrafish embryos were exposed to hydrophobic pollutants and verapamil. Their internal concentrations did not change upon chemosensitisation. Zebrafish embryos were exposed to sediment extracts by passive dosing. Upon chemosensitisation, embryo toxicity increased for one sediment extract. Toxicity remained unaltered or decreased for two other sediment extracts.
a r t i c l e i n f o
a b s t r a c t
Article history: Received 24 April 2017 Received in revised form 30 July 2017 Accepted 5 August 2017 Available online 9 August 2017
Cellular multixenobiotic resistance (MXR) transport proteins enhance the efflux of numerous organic pollutants. However, MXR proteins may be blocked or saturated by xenobiotic compounds, acting as inhibitors d also called chemosensitisers. Although effective on a cellular level, the environmental relevance of chemosensitisers has not been conclusively demonstrated. Since sediments are an important source of bioaccumulating compounds in aquatic ecosystems, sediments and sedimenteassociated hydrophobic pollutants were investigated for their potential to increase exposure and toxicity in the presence of chemosensitisation. In this study, we address this issue by (1) comparing the net uptake of 17 hydrophobic environmental pollutants by zebrafish (Danio rerio) embryos in the presence and absence of the model chemosensitiser verapamil and (2) investigating the impact of verapamil on the dose edependent effect on zebrafish embryos exposed to polluted sediment extracts. None of the 17 pollutants showed a reproducible increase in bioaccumulation upon chemosensitisation with verapamil. Instead, internal concentrations were subject to intraespecies variation by a factor of approximately two. However, a significant increase in toxicity was observed upon embryo coeexposure to verapamil for one of three sediment extracts. In contrast, another sediment extract exhibited less toxicity when combined with verapamil. In general, the results indicate only a minor impact of verapamil on the uptake of moderately hydrophobic chemicals in zebrafish embryos. © 2017 Elsevier Ltd. All rights reserved.
Handling Editor: Jim Lazorchak Keywords: MicroeQuEChERS Multiemode inlet (MMI) GCeMS/MS Large volume injection (LVI) Small volume internal concentration Chemosensitisation Bioaccumulation Sediment toxicology Zebrafish embryo toxicity
1. Introduction With a steadily increasing number of anthropogenic chemicals introduced to the market, environmental pollution has become as
* Corresponding author. UFZ d Helmholtz Centre for Environmental Research, Department of EffecteDirected Analysis, Permoserstr. 15, 04318 Leipzig, Germany. E-mail address:
[email protected] (W. Brack). http://dx.doi.org/10.1016/j.chemosphere.2017.08.019 0045-6535/© 2017 Elsevier Ltd. All rights reserved.
complex, ubiquitous, and diverse as never before (Daughton, 2005). Hence, exposure to xenobiotic compounds is a major stressor for many organisms. Consequently, the toxicokinetic and -dynamic processes, which determine their toxicity in organisms, are among the major concerns in ecotoxicology. While toxicodynamics summarise the modes of action and effects exhibited by pollutants, toxicokinetics, or the concept of absorption, distribution, metabolism, and excretion (ADME), enclose transport processes determining an organism's exposure. In addition to passive diffusion,
D. Kurth et al. / Chemosphere 186 (2017) 652e659
partitioning, and metabolism, active cellular efflux is an important process of the toxicokinetic transport chain (Schwarzenbach et al., 2005; Smital and Kurelec, 1997; Kurelec, 1995). As all those processes interact, resulting in the removal of harmful toxicants from an organism, understanding and predicting the effect of exposure to complex chemical pollution is an inevitable and challenging task. Mixture toxicity research has attracted substantial attention in the last decade (Faust et al., 2001; Backhaus and Faust, 2012; European Commission, 2012). Toxicodynamic models have been developed to predict combined effects in organisms. However, such models can lead to the underestimation of toxicity, especially when biological transport processes are expected to have a major influence (Kurelec, 1997). This has been hypothesised for the substrates of broadlyebinding multixenobiotic resistance (MXR) transport proteins, belonging to the adinosine triphosphate (ATP) binding cassette (ABC) transporter proteins (Kurelec, 1995). Specifically, these proteins are responsible for reducing the concentrations of toxicants in the intraecellular space by translocating them to the extracellular space, where they can be further transported and excreted (Bard, 2000; Aller et al., 2009; Epel, 1998; Kurelec, 1992; Gottesman et al., 2002). However, MXR may be inhibited by environmental pollutants d soecalled chemosensitisers d, which either interfere with the required enzyme activity for the supply of energy as ATP or block the system by slow transport kinetics and/or high binding site affinity (Bard, 2000; Choi, 2005; Ford and Hait, 1994; Kurelec, 1997; Sarkadi et al., 2006). While its importance has been well established on a cellular level, the environmental impact of MXR and its inhibition remains unclear (Kurth et al., 2015). On the one hand, many studies stress the impact of MXR inhibition on bioaccumulation by complex in environmental mixtures (Smital and Kurelec, 1997; Shúilleabha et al., 2005; Zaja et al., 2013; Smital and Sauerborn, 2002). On the other hand, an induction of protein expression overcoming inhibition has been observed in organisms in polluted habitats, for example river sediments (Smital and Kurelec, 1998). Sediments are especially important in ecotoxicology, as they serve as longeterm sinks for environmental pollutants and therefore acquire complex pollution profiles. They have been studied for their MXR inhibitory potential (Zaja et al., 2013), yet less so with respect to the environmental relevance of that potential (Kurth et al., 2015). This relevance depends on both the presence of potent chemosensitisers and the presence of toxic substrates in sediment mixtures. The zebrafish (Danio rerio) embryo is a popular model organism in ecotoxicology and has been used for the assessment of sediment toxicity in the past (Strmac et al., 2002; Kosmehl et al., 2008; Yang et al., 2010; Wu et al., 2010; Kammann et al., 2004). Its advantages include fast development, simple maintenance, high sensitivity, and complexity (i.e. as compared to cellular in vitro tests) (Scholz et al., 2008; Kosmehl et al., 2008). Furthermore, its MXR proteins have recently been characterised and found to be expressed in early life stages (Fischer et al., 2013). This study aims at providing information on the impact of chemosensitisation at the organism level. Does the bioaccumulation of toxic substrates increase with MXR inhibition? If not, could changes in toxicity still occur, for example in result of an augmented energy demand? In order to answer the first question, zebrafish embryos were exposed to artificial mixtures of a wide range of common hydrophobic pollutants, likely to adhere to the sediment compartment. Embryo net uptake upon coeexposure to verapamil was compared to net uptake in the absence of the model chemosensitiser. To prevent the excessive use of zebrafish embryos, bioaccumulation was determined with a small volume method for the extraction and quantification of internal organism
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concentration. This requires sensitive chemical analysis, which can be achieved by exposure to artificial mixtures instead of sediment extracts. To address the second question, zebrafish embryos were exposed to sediment extracts in the presence and absence of verapamil by passive dosing. The impact of chemosensitisation upon embryo teratogenicity was quantified by comparing the resulting doseeresponse curves. Due to their high sensitivity towards chemical pollution, embryos could be passively exposed to sediment extracts, which has the advantage of closely mirroring environmental exposure conditions. 2. Materials and methods 2.1. Chemicals Chemicals were acquired as analytical standards at the highest purity available from Sigma-Aldrich (Munich, Germany), Honeywell (Seelze, Germany), Merck (Darmstadt, Germany), LGC (Teddington, UK), Toronto Research Chemicals (Toronto, Canada), Dr. Ehrenstorfer (Augsburg, Germany), or ABCR (Karlsruhe, Germany). A mixed standard solution was prepared at 10 mg/mL in acetonitrile and stored at 20 C. 2.2. Bioaccumulation experiment Adult zebrafish maintenance and breeding was conducted according to the standard protocol (Westerfield, 1995) and is briefly summarised in the Supplementary Information (SI, Section S1). The collection of eggs and the culturing of embryos was performed as described by Nagel (2001). Briefly, spawning was induced by turning on the light in the morning. Fertilised embryos were collected from glass trays covered with a 3 mm mesh and artificial plants. After rinsing, zebrafish embryos were selected at the 4 to 8ecell stage using a light microscope. Zebrafish embryos were independently exposed to three compound mixtures of narrow log D ranges (see Table 1) in the presence and absence of the chemosensitiser verapamil. Verapamil is a nonespecific inhibitor targeting a variety of MXR proteins €inen and Kukkonen, 2015). Experiments were conducted in (Vehnia 10 mL glass vials with aluminum caps containing 2 mL of ISOewater (SI, Section S1) and ten zebrafish embryos. Each experiment consisted of a noneverapamil solvent control, a verapamilecontaining solvent control, a noneverapamil compound mixture of each log D group, and a verapamilecontaining compound mixture of each log D group. Each group was again subdivided into five exposure replicates per group and five blank replicates, spiked with the respective amount of acetonitrile and methanol. Exposure concentrations amounted to 10 ng/mL (2 mL 10 mg/mL stock solution in acetonitrile), while the verapamil group was additionally spiked with 4 mL 1 mg/mL verapamil (in methanol) to achieve a nominal concentration of 2 mg/mL. Zhu et al. (2014) estimated an LC10 of 166 mg/mL for verapamil hydrochloride in Danio rerio embryos. In order to account for solvent influence, all exposure replicates in the control and both groups received the same amount of acetonitrile and methanol. All embryos were exposed for 72 h on a horizontal shaker at 100 rpm and 26 C. Before exposure termination, embryo lethal, sublethal, and teratogenic effects were recorded (SI, Section S6). Dead embryos were removed from the exposure medium to prevent microbial infestation of healthy embryos and oxygen depletion. Subsequently, embryos were extracted according to a microeQuEChERS (Quick, Easy, Cheap, Effective, Rugged, and Safe) method coupled to a gas chromatography tandem mass spectrometer as detailed in Kurth et al. (2017). Briefly, after thorough rinsing with ISOewater and the manual deechorination of nonehatched embryos, ten embryos
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Table 1 List of analysed compounds, their properties, and detection parameters. Partition coefficients (log D and log Kow) were calculated using the calculator plugeins of JChem (Chemaxon, Budapest, Hungary). TBP: tributylphosphate; TDCPP: tris(1,3-dichloro-2-propyl)phosphate. log D
Compound
Molar mass
log Kow
log D pH ¼ 7.5
[Da]
Retention time [min]
MRM transition 1 m/z
CE
MRM transition 2 m/z
CE
MRM transition 3 m/z
CE
4.0e4.3
TBP Piperonyl butoxide Chlorothalonil Prosulfocarb Diazinone TDCPP Pyrene Carfentrazone-ethyl
266.31 338.21 265.91 251.39 304.35 427.88 202.25 411.04
4.09 4.10 4.10 4.17 4.19 4.28 4.28 4.29
4.09 4.10 4.10 4.17 4.19 4.28 4.28 4.29
11.18 26.65 15.26 17.54 15.12 24.87 21.12 24.09
99.0 / 81.0 176.1 / 103.1 265.9 / 230.9 128.1 / 43.1 137.1 / 84.0 209.1 / 99.0 202.1 / 201.1 326.2 / 177.0
20 25 20 10 15 5 30 20
99.0 / 63.0 176.1 / 131.1 266.1 / 133.0 128.1 / 86.0 137.1 / 54.1 191.0 / 74.9 202.1 / 200.1 325.8 / 121.0
35 15 30 0 15 10 30 60
155.0 / 99.0 176.1 / 117.1 266.1 / 168.0 91.0 / 65.0 152.1 / 137.1 74.9 / 49.0 100.9 / 100.2 330.0 / 310.1
5 20 30 15 0 20 10 15
4.5e4.8
Flusilazole Bifenox Celestolide Galaxolide Triphenylphosphane oxide 2,6-Ditertbutylphenol
315.10 340.99 244.37 258.40 378.09 206.32
4.58 4.63 4.67 4.72 4.76 4.76
4.09 4.63 4.67 4.72 4.76 4.76
22.46 28.37 13.96 16.03 28.61 10.48
233.0 189.1 229.1 243.2 277.0 190.8
/ / / / / /
165.1 126.0 43.1 213.2 199.1 91.0
15 20 25 15 35 35
233.0 189.1 229.2 243.2 276.8 191.1
/ / / / / /
91.0 133.0 131.2 185.2 170.1 163.1
20 15 15 15 60 10
314.7 340.9 229.2 258.2 276.8 191.2
/ / / / / /
232.9 309.9 173.1 243.2 152.1 131.1
10 10 5 10 50 20
5.5e5.8
Dicofol Permethrin 4-Nonylphenol
370.49 390.08 220.35
5.56 5.70 5.74
5.56 5.70 5.74
18.82 32.22 16.63
139.0 / 111.0 183.1 / 168.1 107.1 / 77.0
15 10 20
138.9 / 75.0 183.1 / 165.1 107.1 / 51.1
30 10 30
249.9 / 215.1 183.1 / 153.1 219.8 / 107.1
10 15 20
were transferred into 200 mL glass inserts. In order to obtain four full exposure replicates, coagulated embryos were replaced by replenishing from the fifth exposure replicate. Water volume (ISOewater from embryo transfer) was reduced to approximately 70 mL, 70 mL of acetonitrile were added, and phase separation was induced by the addition of 28 mg of MgSO4 and 7 mg of NaCl. The acetonitrile phase was used for further analysis. Three independent replicate experiments following the above descriptions were conducted on different days. 2.3. Sediment sampling and extraction Sediment samples were collected from the Elbe river basin at three sites: Spittelwasser creek, a minor tributary to river Mulde close to the German industrial area of Bitterfeld, characterised by the strong impact of former chemical industry (5141041.900 N, 12170 20.000 E, May 2008), Bilina, an Elbe tributary with marked petroechemical pollution downstream the city of Most in the Czech Republic (50 300 18.200 N, 13 400 58.500 E, composite sample June 2006/July 2007), and from the River Elbe near Prelouc, a Czech town downstream of a dye and varnish production area (50 020 35.700 N, 15 340 27.600 E, April 2005) (Schwab et al., 2009). After sampling, sediments were freeze-dried and sieved for the silt and clay fraction (
