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

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.