Evaluating Complex Mixtures in the Zebrafish Embryo by Reconstituting Field Water Samples: A Metal Pollution Case Study

Abstract

Accurately assessing the toxicity of complex, environmentally relevant mixtures remains an important challenge in ecotoxicology. The goal was to identify biological effects after exposure to environmental water samples and to determine whether the observed effects could be explained by the waterborne metal mixture found in the samples. Zebrafish embryos were exposed to water samples of five different sites originating from two Flemish (Mol and Olen, Belgium) metal contaminated streams: “Scheppelijke Nete” (SN) and “Kneutersloop” (K), and a ditch (D), which is the contamination source of SN. Trace metal concentrations, and Na, K, Mg and Ca concentrations were measured using ICP-MS and were used to reconstitute site-specific water samples. We assessed whether the effects that were observed after exposure to environmental samples could be explained by metal mixture toxicity under standardized laboratory conditions. Exposure to “D” or “reconstituted D” water caused 100% mortality. SN and reconstituted SN water caused similar effects on hatching, swim bladder inflation, growth and swimming activity. A canonical discriminant analysis confirmed a high similarity between both exposure scenarios, indicating that the observed toxicity was indeed primarily caused by metals. The applied workflow could be a valuable approach to evaluate mixture toxicity that limits time and costs while maintaining biological relevance.

Advancing the Zebrafish embryo test for endocrine disruptor screening using micro‐injection: Ethinyl estradiol as a case study

“…To avoid a time‐dependent effect (i.e., age of larvae increasing during the day of measurement), all heart rate measurements were performed within 1 h starting at 24 hpf, and measurements were alternated randomly among exposure conditions. Morphological scoring involved assessment of 28 binary parameters (Supplemental Data, Table S2; Michiels et al ). All embryos were analyzed for swimming activity at 72, 96, and 120 hpf in a behavioral tracking instrument (Zebrabox 3.0; Viewpoint) for 50 min under normal light conditions (100% light setting, 1160 lux).…”

An AOP-based alternative testing strategy to predict the impact of thyroid hormone disruption on swim bladder inflation in zebrafish

“…A prediction model based on only DIO1 and DIO2 enzyme inhibition data would therefore not be able to predict these effects (i.e., generate a false negative prediction). In addition to the fact that differences in predicted effects may be observed as a result of, for example, specific toxicokinetic and/or toxicodynamic properties, and taking into account that effects in fish can possibly be more accurately predicted using fish tissue-based assays rather than using porcine tissue, it is also highly plausible that many different toxicological mechanisms can lead to swim bladder inflation effects (Hagenaars et al, 2014;Li et al, 2011;Michiels et al, 2017;Sarnowski, 2004;Trotter et al, 2003;Villeneuve et al, 2014;Woolley and Qin, 2010;Yin et al, 2011). Some of these mechanisms may even be related to the hypothalamic-pituitary-thyroid (HPT) axis and disruption of TH balance but currently not be captured by our AOP network and in chemico assays (e.g., at the level of the sodium-iodine symporter, the production of thyroid stimulating hormone, etc.…”

“…The publication of OECD Testing Guideline (TG) 236, the "Fish Embryo Acute Toxicity (FET) Test" (OECD, 2013a), describing a 96 h fish embryo test, has greatly facilitated the use of fish embryos in toxicity studies. The testing guideline is currently limited to observations of lethal endpoints and hatching, but research has shown that more subtle toxic effects can also be reliably investigated using fish embryos (Braunbeck et al, 2014;Hagenaars et al, 2014;Hill et al, 2005;Michiels et al, 2017;Pype et al, 2015;Scholz et al, 2008;Selderslaghs et al, 2013;Stinckens et al, 2016;Verstraelen et al, 2016;Voelker et al, 2007). However, the development of alternative assays capable of capturing and representing the mechanisms underlying toxicity pathways at sub-organismal levels of biological organization requires a targeted approach.…”

Temperature Effects During a Sublethal Chronic Metal Mixture Exposure on Common Carp (Cyprinus carpio)

“…Moreover, in Flanders (the Belgian region where this study was conducted), the limits for these metals are set to 0.11 μM for Cu and 0.30 μM for Zn, whereas for dissolved Cd in rivers and lakes limits range (according to the water hardness) are between 0.004 to 0.013 μM (or 0.45 and 1.5 μg/L) ( VLAREM II, 2010 ). However, these limits can easily be exceeded as shown by Michiels et al (2017) , who reported dissolved metal concentrations up to 0.95 μM, 0.05 μM, 52 μM for Cu, Cd, and Zn, respectively, in two different rivers. In addition a more recent study reported values ranging, from 0.02 to 0.04 μM for Cu, from 0.74 to 52 μM for Zn, and from 0.004 to 0.06 μM for Cd over five different locations in Belgium ( Delahaut et al, 2019 ).…”