Hydrogen Peroxide Treatment in Atlantic Salmon Induces

19 Daily variation in the absorption, metabolism and excretion of toxic substances will 20 ultimately determine the actual concentration to which the cells and tissues are exposed. In 21 aquaculture, Atlantic salmon (Salmo salar) can be frequently exposed to hydrogen peroxide 22 (H 2 O 2) to treat topical skin and gill infections, particularly in relation to parasitic infections 23 (e.g. sea lice Lepeophtheirus salmonis and amoebic gill disease caused by Neoparamoeba 24 perurans). It is well accepted that the time of administration influences pharmacodynamics 25 and pharmacokinetics of drugs which in turn affects their efficacy and toxicity. Consequently, 26 a better understanding of drug side effects as a function of time of day exposure would help 27 to improve treatment efficacy and fish welfare. To this end, salmon were exposed to H 2 O 2 28 (1500 mg/L) for 20 min at six different times of the day during a 24-h cycle and we 29 investigated the time-dependent effects of exposure on physiological stress (glucose, lactate 30 and cortisol) and antioxidant enzyme expression (gpx1, cat, Mn-sod and hsp70) in liver and 31 gills. In addition, at each sampling point, 8 control fish were also sampled. Our results 32 revealed that the time of administration of H 2 O 2 caused significant differences in the 33 induction of both physiological and oxidative stress responses. Glucose and lactate were 34 higher in the treated fish during daytime whereas cortisol levels appeared to be systematically 35 increased (>1000 ng/mL) after H 2 O 2 treatment irrespective of exposure time, although 36 differences with control levels were higher during the day. In liver, gene expression of 37 antioxidant enzymes displayed daily rhythmicity in both treated and control groups and 38 showed higher mRNA expression levels in salmon treated with H 2 O 2 at ZT6 (6 h after lights 39 onset). In gills, rhythmic expression was only found for gpx1 in the control fish and for hsp70 40 and Mn-sod in the treated groups. However, in the treated salmon, higher gene expression 41 levels of all the investigated enzymes were also observed at ZT6-10. Clock gene expression 42 showed rhythmicity only in the liver in accordance with the daily rhythm of enzyme 43 expression observed in this tissue. Altogether, this study provides first evidence of 44 chronotoxicity in Atlantic salmon treated with H 2 O 2 and suggests increased sublethal toxic 45 effect …