In: G.M.Hallegraeff, S.I.Blackburn, C.J. Bolch & R.J.Lewis (eds), Harmful Algal Blooms 2000. Proc. 9th Int. Conf.Harmful Algal Blooms. IOC. Paris 2001. MITIGATION BY CYSTEINE COMPOUNDS OF RHEOTOXICITY, CYTOTOXICITY AND FISH MORTALITY CAUSED BY THE DINOFLAGELLATES, GYMNODINIUM MIKIMOTOI AND G. CF. MAGUELONNENSE. Ian R. Jenkinson* and GeneviLve Arzul** *ACRO, Lavergne, 19320 La Roche Canillac, France. ian.jenkinson@wanadoo.fr and Stazione Zoologica ’Anton Dohrn’ di Napoli, Villa Comunale, 80121 NAPOLI, Italy **IFREMER, Centre de Brest, B.P. 70, 29280 PlouzanØ, France. genevieve.arzul@ifremer.fr ABSTRACT Two Gymnodinium species were studied for their rheo- toxic and cytotoxic activity. G. mikimotoi was found to have marked rheotoxicity and relatively low cytotoxicity (measured as haemolytic activity), while G. cf. mague- lonense showed both effects. The effects of cysteine compounds, known as mucolytic agents and as protectors against O-radical damage, were investigated as a possible mitigation tool. N-acetyl-L-cysteine and ethyl-L-cysteine ester reduced: fish mortality; rheological yield stress; haemolytic activity. Beneficial action started at 0.01 mMolar. L-cysteine is also active. The study suggests that addition of cysteine or its compounds to the water may be an economically feasible mitigation tool. INTRODUCTION Some phytoplankton secrete polymers that make the water more viscous [1, 2]. This effect can asphyxiate fish by reducing water flow and hence oxygen availability at their gills [3], when it is termed rheotoxicity. Some exosecretions of harmful phytoplankton are also associ- ated with cytotoxicity, which can be assayed by meas- uring their haemolytic activity [3,4,5]. The aim of the present study has been to apply the results of previous studies on the rheology [6] and other ichthyotoxic [7,8] properties of seawater including its phytoplankton, so as to mitigate the total toxicity to fish during HABs of two ichthyotoxic Gymnodinium species. To assay toxicity, we used the survival time of fish. Both Gymnodinium species showed haemolytic activity, while the rheology of the water constituted a second indicator of its biological quality in relation to fish survival. We will show that the use of mucolytic substances, derivatives of cysteine [9, 10, 11], allowed the toxic properties to be markedly reduced in both cases. MATERIAL AND METHODS The culture of Gymnodinium mikimotoi (G. mik) studied (Stock GM95TIN) had been isolated at Tinduff (Rade de Brest, France) in 1995, while that of G. cf ma- guelonense (G. mag), isolated by E. Erard-Le Denn, originated from Tunisian waters, north of Sfax in the Gulf of GabLs in 1994, after an intensive fish kill. Both cultures were cultivated at 18C, and illuminated 12h/12h at 60mole quanta.m -2 .s -1 at IFREMER, Brest. Enriched f/2 medium was used, made using ocean water of salinity 36 ppt. All cultures were investigated at the end of their exponential phase. Cells were enumerated by optical microscopy. Seabass, Dicentrarchus labrax, of about 80 g, reared under controlled conditions on untreated feed by the Department of Living Resources, IFREMER, were used for investigation both of survival and of flow through the gills. Fish survival tests were carried out on batches of three fishes in 3 litres of algal culture or filtered seawater under a battery of fluorescent lamps. Investigations were furthermore done with and without aeration, so that in- formation about the effect of the oxidative environment on fish survival might help elucidate any role of free oxy- gen species associated with algal toxicity. Survival time was taken as that until the fish turned ventral side up for the first time. Survival time in seawater without aeration was considered to be limited by available oxygen content. Haemolytic activity in cultures was determined using the method of Arzul et al. [12], slightly modified to ob- tain a more sensitive repsonse. This test reveals the pres- ence of substances able to lyse cell membranes. We used horse red blood cells (RBCs) from Pasteur-Sanofi, Paris, or human RBCs when horse RBCs were unavailable. Some G. mik culture was subject to sonification using a Deltasonic fi apparatus for 8 minutes at 42 kHz (Table 2b). Rheological measurements of flow through fish gills under differing hydrostatic pressure differences were made following Jenkinson & Arzul [3]. The same gill- flow apparatus was used. We estimated the yield stress y of the culture or seawater, by solving for y in the equation y e y P P n k t + - = ′ - ) / ( 0 ). ( where P 0 and P t are the hydrostatic pressure at times 0 and t respectively. η and k (both assumed to remain con- stant during an individual flow trial) are the dynamic viscosity (in the limit of high shear rate) and k is a flow coefficient for the geometry of the gill passages. The L-cysteine (C), ethyl L-cysteine ester (EC) and N-acetyl-L-cysteine (AC) were provided by Sigma and used as dilutions of stock solutions in seawater. RESULTS Effects of aeration, AC and EC on fish survival in Gymnodinium culture We made a first batch of experiments to investigate the effects of aeration and EC. In this series of experi- ments, the G. mik was of concentration 21 million cells L -