## 3 Case Study: Effect of Household Cleaning Agents on Zebrafish Swimming Speed  
In order to demonstrate the usefulness of AniWellTracker, zebrafish were exposed to two types of household cleaning agents, and imaging was performed, followed by analysis with AniWellTracker. The household cleaning agents, a baking soda cleaner (Cleaner BS) and a bathroom cleaner (Cleaner BC), were purchased from a local market in S. Korea. Wild-type zebrafish were obtained from the Korea National Research Resource Center and mated to obtain eggs, after which they were grown at 28 \({}^{\circ}\)C. In the experiment, 18 days post-fertilization (dpf) fries were used. A total of 990 \(\mu\)L of the breeding water was placed in a 15-well spot plate (FLINN Scientific, item #: AP6404), and one zebrafish larva \begin{table}
\begin{tabular}{c c} \hline \hline
**Output File Name** & **Explanation** \\ \hline Tracking\_AbsoluteLocation.csv & Animal’s centroid (x and y) in the image \\ Tracking\_RelativeLocation.csv & Animal’s centroid (x and y) within the ROI \\ Tracking\_TravelDistance.csv & Animal’s travel length between time-lapse images \\ Tracking\_TravelSpeed.csv & Animal’s travel speed between time-lapse images \\ Tracking\_CentralAngle.csv & Animal’s central angle between the center of the ROI and the location of the animal \\ Tracking\_DistanceFromCenter.csv & Distance between the center of the ROI and the centroid of the animal \\ Tracking\_RotationAngle.csv & Change in the central angle of the animal between time-lapse images \\ Tracking\_ & \\ TravelDistance\_TimeBlock00 min.csv & Average distance traveled in each time block of 00 min where 00 represents a number \\ Tracking\_ & \\ TravelSpeed\_TimeBlock00 min.csv & Average speed traveled in each time block of 00 min where 00 represents a number \\ Tracking\_LocationHeatmap.csv & Heatmap for the number of places an animal was located in the ROI \\ Tracking\_ & \\ DistanceFromCenterHeatmap.csv & Heatmap of the number of distances the animal was located from the center of the ROI \\ \hline \hline \end{tabular}
\end{table} 
Table 1: Analysis output files generated by AniWellTracker. was placed in each well. Then, 10 uL of diluted cleaner was added, after which imaging was performed. Animal experiments using zebrafish were approved by the Peachchem Animal Care and Use Committee (IACUC) under a protocol (Peachchem P-20190507-1). The zebrafish images were captured at 1 s intervals using the time-lapse capture function of PhenoCapture from a custom imaging device equipped [24] with a Logitech C920 camera and a computer (Intel Core i3 2.27 GHz processor; 4 GB ram; Microsoft Windows 10). The equipment was covered with blackout curtains to exclude the influence of external lighting. After imaging for a total of about 2 h, the animal's position was analyzed using the tracking function of AniWellTracker. Regarding the AniWellTracker's animal tracking performance, it took an average of 1.38 \(\pm\) 0.02 s to process one 1920 \(\times\) 1080 JPEG image on an office PC (Intel Core i7-8700 3.20 GHz processor; 16 GB ram; Samsung SSD 512 GB; Windows 10). The speed as a function of time for the two household cleaners is shown in Figure 5A. In Cleaner BS (baking soda cleaner), the animals showed movement for approximately 60 min, and barely any movement thereafter; thus, they were estimated to have died after approximately 60 min. Indeed, after 2 h of image capture, the animals were confirmed to have died. In comparison, in Cleaner BC (bathroom cleaner), the animals showed active movement during the 2 h experiment, indicating that Cleaner BS was likely more toxic than Cleaner BC. In Figure 5B, the unit of speed is pixel/sec; to compare with other experiments, the unit must be changed to cm/s. To convert the distance from units of pixels to centimeters, a conversion factor is calculated. This can be used to obtain the conversion factor and measure the actual distance (cm) between specific points of the image easily. The overall average speed (cm/s) under test conditions is shown in Figure 5C.