Article pubs.acs.org/est
Toxicokinetics of Polar Chemicals in Zebrafish Embryo (Danio rerio): Influence of Physicochemical Properties and of Biological Processes Stephan Brox,† Bettina Seiwert,† Eberhard Küster,‡ and Thorsten Reemtsma*,† †
Department of Analytical Chemistry, Helmholtz Centre for Environmental Research - UFZ, Permoserstrasse 15, 04318 Leipzig, Germany ‡ Department Bioanalytical Ecotoxicology, Helmholtz Centre for Environmental Research - UFZ, Permoserstrasse 15, 04318 Leipzig, Germany S Supporting Information *
ABSTRACT: The time-resolved uptake of 17 nonionic and ionic polar compounds (logD ≤ 2) with a diversity of functional groups into zebrafish embryos (ZFE) was studied over 96 h of exposure. Among them were pharmaceuticals, pesticides and plant active ingredients. Uptake rates for the diffusion controlled passive uptake through the ZFE membrane ranged from 0.02 to 24 h−1 for the nonionic compounds and were slower for ionic compounds (90% of the initial amount taken up into the ZFE. For six compounds internal concentrations remained very low (rel. int. conc. < 0.2). Besides biotransformation (sulfamethoxazole), poor membrane permeability (cimetidine, colchicine) and also affinity to efflux transporters (atropine and chloramphenicol) are the likely reasons for these low internal concentrations. This study outlines that the uptake of polar compounds into ZFE is influenced by their physicochemical properties. However, biological processes, biotransformation and, likely, efflux can strongly affect the internal concentrations already in early developmental stages of the ZFE. This should be considered in future toxicokinetic modeling. The evaluation of the toxicity of chemicals by ZFE requires toxicokinetic studies of the test compounds and their TPs to increase comparability to effects in fish.
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INTRODUCTION The zebrafish (Danio rerio, Hamilton-Buchanan, 1822) embryo (ZFE) is widely used as a test system in ecotoxicology and environmental risk assessment.1,2 Due to its small size and transparency it can be applied in high-throughput studies recording lethal effects,3,4 developmental effects5,6 or behavioral change.3 Today the ZFE is partially seen as an alternative to toxicity testing with juvenile or adult fish.7−9 To better understand the strength of biological effects of chemicals on the ZFE toxicokinetic (TK) and toxicodynamic (TD) processes at different life-stages of the ZFE need to be considered since these processes affect the internal and target concentrations, i.e. the internal dose. The ZFE develops fast during the test duration of 100 h post fertilization (hpf): major organs are differentiated within the first 72 hpf, liver and bile cells are visible after 72 hpf10−12 and its pool of enzymes is also developing rapidly.13−15 Correspondingly, drastic changes may be expected for the rate of uptake, distribution, metabolism (here denoted as “biotransformation”), and excretion (ADME) of test chemicals by the ZFE during this period of exposure. © 2016 American Chemical Society
Unlike other higher aquatic organisms utilized for toxicity testing, such as fish or daphnia, the ZFE is not developed enough to perform uptake via its gills or the gastrointestinal tract, because in its embryonic life-stages these organs are not fully developed and operational. Thus, uptake (as well as excretion) of test compounds can only occur through the ZFE membranes, primarily by diffusion through its comparatively large surface area.16,17 For certain compounds also carrier-mediated uptake through the ZFE membrane may occur but its impact on the internal concentration has still to be evaluated.18,19 One can assume that due to the limitations of diffusion as the sole mechanism of uptake into the ZFE, uptake rates could be significantly slower than for test organisms with fully developed gastrointestinal tract and gills. Moreover, physicochemical properties of the test compounds may be more crucial for the uptake into ZFE. Considering that uptake is a prerequisite for the development of effects and that the test duration of the ZFE test Received: August 25, 2016 Accepted: August 29, 2016 Published: August 29, 2016 10264
DOI: 10.1021/acs.est.6b04325 Environ. Sci. Technol. 2016, 50, 10264−10272
Article
Environmental Science & Technology is limited to a maximum of 120 hpf20 there is a strong need to closely study the uptake kinetics of test compounds into the ZFE. This is also essential for the comparison of toxic effects with other test organisms.21 For toxicity testing the time-resolved observation of effects on the ZFE for a period of 96 h is recommended22 and should be accompanied by the determination of the internal concentration for the study compound. Although the internal concentration of test compounds has been determined in an increasing number of studies, most of them focused on lipophilic test compounds23−28 and on somewhat older life-stages of the ZFE (>72 hpf).23,24,27−30 To date, studies comparing the time-resolved uptake of a large number of structurally diverse polar test compounds under similar exposure conditions starting in very early life-stages of the ZFE (50%) in the results of the first two independent experiments. In those cases mean values are obtained from nine replicates per sampling point (n = 9). In a few cases an outlier test (Grubbs) was performed and, when positive, the respective data point was excluded from the calculation of mean values and standard deviation. Normal distribution of the data points was checked and confirmed before. Data for theophylline were obtained from one preliminary experiment with five replicates per sampling point in which ZFE of
