DanioFlow – Automated Behavioral Analysis Pipeline for Zebrafish Larvae

Version 2 introduces a fully updated DanioFlow workflow including automated Benchmark Dose (BMD) modeling (Hill model), integrated summary sheet generation, and behavioral fingerprint classification.

All files have been updated to reflect the new analysis pipeline and reporting outputs.

DanioFlow is an automated behavioral analysis pipeline for high-throughput zebrafish (Danio rerio) larval assays in toxicology and neurobehavioral research. The tool processes raw locomotion tracking data exported from EthoVision (or equivalent tracking software), applies multiple quality control filters, performs statistical analyses, and generates standardized Word reports with interpreted results. DanioFlow is implemented as a Microsoft Excel workbook with embedded formulas, macros, and reporting functionality, enabling non-programmers to perform complex analyses without specialized coding skills.

DanioFlow supports two widely used sensory stimulus paradigms: Light vs. Dark and Tapping vs. Silence, and is optimized for experiments performed in 96-well plates (but could be adapted to other plate formats) with predefined dose–response rows and control conditions. The workflow includes:

• Import of raw tracking data

• Automated filtering of low-quality detections

• Removal of low-mobility and IQR outliers

• One-way ANOVA with Fisher’s LSD post-hoc tests

• Benchmark Dose Modeling using embbeded Macro

• Automatic graph generation and tabular summaries

• Export of integrated results to Microsoft Word (.docx)

Multiple internal validation and quality control steps ensure robustness of the behavioral data, including tracking completeness thresholds, movement thresholds, plate control validation, and stimulus-specific consistency checks.

DanioFlow is intended to support academic, governmental, and industrial research applications involving zebrafish larval behavior, neurotoxicity screening, environmental risk assessment, and chemical hazard profiling. It enables efficient processing of behavioral datasets while preserving transparency in data filtering, statistical assumptions, and reporting structure. While DanioFlow streamlines the analysis workflow, expert interpretation remains essential for regulatory or publication use.

Keywords: zebrafish, toxicology, behavior, neurobehavior, EthoVision, Danio rerio, anxiety, thigmotaxis, BMDS, dose-response

Funded by Horizon Europe. PARC-HORIZON-HLTH-2021-ENVHLTH-03 - Project: 101057014.

How to cite:  Torres-Ruiz, M. DanioFlow (vx.x): Automated behavioral analysis pipeline for zebrafish larvae. (Software), 2025. http://doi.org/10.5281/zenodo.18249245