Background: Dichlorooctylisothiazolinone (DCOI) is a replacement wood preservative for pentachlorophenol and a marine anti-fouling agent. In August 2023, a DCOI mixture spill occurred at Stella-Jones in Sheridan, Oregon, prompting monitoring by the Oregon Department of Environmental Quality (DEQ). Study: Following the spill, a single static neurobehavioral experiment on zebrafish embryos was performed; however, DCOI is known to degrade in aquatic matrices. This study aimed to: 1) measure DCOI in surface waters adjacent to Stella-Jones; and 2.) repeat the zebrafish neurobehavioral experimental study at low doses and daily exposure renewals. At 120 hours post fertilization (hpf), larval photomotor response (LPR) was measured. Results: DCOI was not detected in the six surface water samples collected (< 0.01 µg/L), suggesting that releases do not occur daily, or may occur sporadically. Statistically significant hyperactivity was observed in the LPR dark phase and statistically significant hypoactivity was observed in the LPR light phase. Neurobehavioral impacts may be an important outcome from DCOI exposure; however, further investigation is needed to determine whether the LPR behavior deficits manifest as abnormal motor, social, or cognitive behavior deficits in adult zebrafish.