Hydrogen peroxide in seahorse aquaculture: Determining safe exposure levels using non-invasive biomarkers of stress

Hydrogen peroxide (H 2 O 2 ) is widely used in aquaculture to prevent and control fish mortality often associated with fungal, bacterial, and parasitic infections. Its main advantage by its reduced environmental impact. However, H 2 O 2 affects redox regulation of cell survival and proliferation, controls inflammatory and immune responses, and is related to oxidative stress. Here we evaluated the behavioral response, opercular beats, and weight loss as stress indicators in captive-bred Hippocampus reidi seahorses exposed to four different concentrations of H 2 O 2 (4.4 mM, 10.9 mM, 12.0 mM, and 13.0 mM). The lowest concentration (4.4 mM) was considered safe up to 60 min of exposure, with little impact on weight loss, behavior changes, and opercular beats. Conversely, H 2 O 2 concentrations ≥10.9 mM led to a high frequency of stress-associated behaviors (irregular swimming, pipe, and curled tail), severe alterations in locomotion and opercular beats, and were lethal at 12 mM and 13 mM. The lowest dose tested increased the locomotion time of seahorses by 55% and decreased benthic behavior by 47% compared to seahorses not exposed to H 2 O 2 . The opercular beats were increased in the presence of H 2 O 2 following a dose-response curve. H 2 O 2 exposure induced a significant decrease in the mean body weight of the seahorses in a dose-dependent manner. We conclude that H 2 O 2 can be used as a potential treatment by immersion (i.e. bath) for H. reidi in a concentration of 4.4 mM for 60 min. H 2 O 2 concentrations and its potential use in immersion treatment (bath) for Hippocampus reidi. • Behavioral shifts and opercular beats are reliable indicators of toxicity. • Seahorses displayed four exclusive stress behaviors under high H 2 O 2 concentrations. • H 2 O 2 -induced decrease in seahorse means body weight in a dose-dependent manner. • A H 2 O 2 concentration of 4.4 mM for 60 min was considered safe for seahorses. • We encourage further research involving safe dose efficiency in H. reidi diseases.