camp. ~iochem. ~~~s~~. Vol. IolC, No. 2, pp. 331-336, 1992 Printed in Great Britain 03~-#92/92 $5.00 + 0.00 Q 1992 Pergamon Press plc zyxwvut ACUTE TOXICITY OF OZONE AGAINST MORPHOLOGY OF GILL AND ERYTHROCYTES OF JAPANESE CHARR (SAL ~~LI~U~ L~~C~~A~~rS) KENJI FUKUNAGA,* TETSUYA SUZUKI,*~ MASANORU ARITA,*~ATORU SUZUKI,* AKIHIKO HARA,* KOHEI YAMAUCHI,* NARIKO SHINRIKI,~ Kozo ISHIZAKI,$ and Kozo TAKAMA* *Faculty of Fisheries, Hokkaido University, Hakodate, 041 Japan; and fHokkaido Industry Development Experimental Station, Ministry of Industry and Trade, Sapporo 001, Japan (Tel.: 81-138-41-0131 Ext. 274; Fax: 81-138-43-5015) (Received 23 April 1991) Abstract-l. Acute toxicity of ozone exposure to Japanese charr (Sa/ue[inus leucomaenis) was studied histopathologically and hematologically on gill tissue and red blood cells (RBC) under different ozone conentrations (O-O.7ppm). 2. Exposure of ozone above 0.7 ppm led to characteristic symptoms and all died of choking in 30 min. 3. Many swollen RBC were seen under the scanning electron microscope. 4. RBC congestion was serious in the gill where degeneration of lamellar epithelium was observed. However, injury to chloride cells was not clear. INTRODUCTION Except for those organisms that are especially adapted to live under anaerobic conditions, oxygen is an essential element for all animals, plants and micro- organisms to produce efficient energy production. Oxygen is the very source of the development of life on Earth. However, in some cases oxygen is toxic to organ- isms. A great number of studies on the toxic effect of oxygen have been made, however, they have mostly been studies on biological, chemical, physiological, and clinical aspects. Although studies on the toxic effect of reactive oxygen to organisms at molecular level have also been reported, proper explanation on the mechanism of oxygen toxicity has not yet been made. Ozone, although not strictly an active oxygen species, has a strong oxidation potential. Therefore, some scientists regard it as one of active oxygen. Ozone has been used for disinfection of aquaculture systems for more than 20 years mainly in western countries on a co~ercial basis. Ozone is now used for disinfection of drinking water (Harris, 1974), sewage treatment (Nebel et al., 1973), and as a substitute disinfectant for chlorine (Siegrist et al., 1975; Blogoslawski et al., 1976). Ozone has been studied from the scientific interest as well (Blogoslawski, 1977). As it is well known, ozone shows hazardous effect at higher concentration. Since ozone has been used for pest control by taking advantage of its strong oxidation potential, its usefulness is regarded as the substitute for malachite green in aquaculture for incubation of fish eggs to minimize their loss due to infection caused by Saprolegnia (Benoit and Matlin, 1966), by bacterial pathogens (Wedemeyer et al., 1977), and by viruses (Wedemeyer, 1978). ~To whom all the correspondence should be mailed. As indicated by Wedemeyer et al. (1979a), the adverse effect of ozone in terms of acute and chronic toxicity to fishes must be assessed before introducing it as a routine preventive measure against infection. To give appropriate assessment, basic studies on the biological effect especially from reactive oxygen are essential. There are a considerable number of studies on the effect of ozone on fish (Wedemeyer et al., 1979b; Tipping, 1988; Meldrim, 1988). They have been done mainly from the practical point of view of using ozone for the sterilization of water and/or ~sinfestation of infectants to salmonids. However, in contrast to the practical studies, basic studies on the action mechanism of ozone to fish at the cellular level are comparatively few. Richardson et al. (1983) reported the effects of ozone-produ~d oxidant (OPO) on adult white perch (Morane americana) in estuarine water. On the other hand, many aspects especialy from the biochemical view points including active oxygen scavenging systems and membrane lipid and protein alteration have remained unrevealed, far less than those of other reactive oxygen studies. In the present paper, we examined what and how acute ozone exposure caused on fish by using Japanese charr (SffZue~~~us ieue~~ae~~s) as an experimental fish to observe behaviQra1, hematological and histo- pathological changes. This first step fundamental observation is essential to interpret how the ozone as one species of active oxygen species acts on the biological system. MATERIALS ANII METHODS Fish Fish used in the present study were disease free one year old Japanese charr (Salvelinus leucomaenis) hatched in fall CBPC 101/2---f