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The zebrafish ( Danio rerio) has become an increasingly popular model organism for the study of the gut microbiome, especially in the context of exposure science 1. We found that inclusion of PCR replicates had the smallest effect on our final interpretations, and the effects of dissection method and DNA extraction kit had significant effects in specific contexts, primarily in the cases of identifying microbial biomarkers. pooled triplicate reactions) affected our interpretation of how exposure influences the diversity and composition of the gut microbiome, as well as our ability to identify microbiome biomarkers of exposure. whole fish), three DNA extraction kits (Qiagen Blood & Tissue, Macherey–Nagel NucleoSpin, and Qiagen PowerSoil), and inclusion of PCR replicates (single vs. Using a model toxicant, benzopyrene (B aP), we assessed how each of two dissection methods (gut dissection vs. To guide future investigations using this model system, we defined how various experimental decisions affect the outcomes of studies on the effects of exogenous exposure on the zebrafish gut microbiome. This is especially the case for microbiome investigations in the zebrafish ( Danio rerio) model system, which is quickly becoming a workhorse system for understanding the exposure-microbiome-physiology axis. Rapidly growing fields, such as microbiome science, often lack standardization of procedures across research groups.