Author Institution: Department of Zoology, The Ohio State University, Columbus, Ohio 43210A series of tests with malachite green, used at various concentrations and treatment durations, were made on six species of watermolds known to be parasitic on fish and/or fish eggs. The tests indicate that these species have markedly different tolerances to the dye. While a treatment of as little as 1 ppm of malachite green for five minutes was effective for Saprolegnia parasitica, it took 10 ppm for five minutes to control Achlya ambisexualis and Allomyces macrogynus, and 15 ppm for one hour to control Achlya oblongata

Martin, Richard

The Ohio Journal of Science

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THE EFFECT OF MALACHITE GREEN AS A FUNGICIDERICHARD MARTINDepartment of Zoology, The Ohio State University, Columbus, Ohio $210ABSTRACTA series of tests with malachite green, used at various concentrations and treatmentdurations, were made on six species of watermolds known to be parasitic on fish and/orfish eggs. The tests indicate that these species have markedly different tolerances to thedye. While a treatment of as little as 1 ppm of malachite green for five minutes was effec-tive for Saprolegnia parasitica, it took 10 ppm for five minutes to control Achlya ambi-sexualis and Allomyces macrogynus, and 15 ppm for one hour to control Achlya oblongata.INTRODUCTIONThe occurrence of parasitic fungi constitutes a chronic problem in fish hatcheriesand aquaria. Fish suffering from stress and injury may become infected by fungi,resulting in considerable economic loss, particularly where valuable aquarium andcultured fish are involved. Nigrelli (1943) reported that over 45 deaths in oneyear were incurred among fishes at the New York Aquarium due to infestationsby unidentified species of the genus Saprolegnia. Many fish hatcheries have seriousproblems with fungal infections of fish and fish eggs (Glen L. Hoffman, 196 Xpersonal communication).The most common treatment for parasitized fish and fish eggs is malachitegreen oxalate. This aniline dye is used as a dip or flush to eliminate or preventthe growth of fungi. Many studies have been made to try and determine themost effective concentrations for fungicidal-fungistatic effects. Prominentamong them is that of O'Donnell (1941), who found that a 10-30-second dip in67 ppm (1:15,000) malachite green was effective and non-toxic to 18 species offish. Johnson et al., (1955) found that semi-weekly treatments of malachitegreen at 5 ppm for one hour eliminated losses among fry and eggs due to fungi.It is known that the members of at least six genera of "watermold" fungi arenatural parasites of fish and/or fish eggs (Scott and O'Bier, 1962), includingSaprolegnia, Achlya, Aphanomyces, Pythium, Leptomilus, and Allomyces. It ispossible, however, that there are differences in resistance to malachite greenamong various species within these genera. It was the purpose of this investigationto examine the usefulness of malachite green as a fungicide to six species of fungiand to assay its relative toxicity to them.MATERIALS AND METHODSSix species of fungi, differing in their ability to kill or damage fish were tested.These species were Saprolegnia parasitica, S. ferax, Achlya oblongata, A. racemosa,and both male and female strains of A. ambisexualis, all of which belong to theSaprolegniaceae, and Allomyces macrogynus of the Blastocladiaceae.The seven kinds of fungi were tested with malachite green in the followingmanner. Sterile petri dishes containing 20 ml of 1, 5, 10, and 15 ppm solutionsof malachite green were used in each test. A sesame seed bearing a flourishinggrowth of mycelium with zoosporangia was placed into the dish of test solutionfor 5, 15, and 30 minutes, and 1, 12, and 24 hours. The fungal colony was thenremoved and rinsed twice with double distilled water before being placed in a newpetri dish containing charcoal-filtered tap water and a number of boiled sesameseeds. The test fungi were incubated for seven days in the new petri dish at acontrolled temperature of 22°C and under light conditions of 15 hours of indirectlight alternating with nine hours of darkness during each 24-hour period, in aneffort to simulate natural conditions. If, after seven days, the fungus had notinfested the new seeds, the test was terminated and the time/concentration con-sidered to be effective. Each test was duplicated.THE OHIO JOURNAL OF SCIENCE 68(2): 118, March, 1968.No. 2 FUNGICIDAL EFFECT OF MALACHITE GREEN 119RESULTSThe results of these duplicate experiments to determine the effectiveness ofmalachite green as a fungicide against the seven selected watermolds are sum-marized in Table 1. From this it is apparent that the effects of this chemicalwere different for different species of fungi.Saprolegnia parasitica was prevented from infesting new seeds by as little as1 ppm for five minutes. Saprolegnia ferax showed similar results, although aslightly higher concentration of malachite green—5 ppm—was necessary to preventsubsequent growth on new sesame seeds; it was still capable of propagation whentreated with only 1 ppm of malachite green for five minutes. Achlya oblongatawas the most resistant to malachite green of all species of fungi studied and con-tinued to be capable of propagation of new seeds until the treatment was increasedto 15 ppm for one hour. Achlya racemosa was effectively prevented from propaga-tion by treatment with 5 ppm for five minutes. Achlya ambisexualis (both sexes)and Allomyces macrogynus became incapable of propagating on new seeds afterimmersion in 10 ppm of malachite green for five minutes.TABLE 1Growth of fungus after treatment with malachite green at indicated combinationsof concentration and exposure timeSaprolegnia parasiticaS. feraxAchlya oblongataA. ambisexualis (m)A. ambisexualis (f)A. racemosaAllomyces macrogynusSaprolegnia parasiticaS. feraxAchlya oblongataA. ambisexualis (m)A . ambisexualis (f)A. racemosaAllomyces macrogynusTimeppmTimeppm1OXXXXXX10oXXXoX5 min.500XX00X15O0X00001000X0000hr1000X00001500X00001500o00o0100XXXXX100X0X0X15 min.500X000012 i5O0X00001000X0000hrs10000000o150OXo0o015OOO0ooo1o0XXXXX1ooX0XoX30 min.50OXOO0O2450OX00O01000X0000hrs10oo00o0o15OOX00oo15OOO0oooControls used in each test exhibited growth in from 48-72 hours.Symbols: O—no growthX—growth within 7 days(m)—male (f)—femaleEach test was duplicated.DISCUSSIONFish and fish eggs are probably not immune to infection, with exposure to15 parts of malachite green oxalate for only up to 30 minutes, because the testsindicate that at least one species, Achlya oblongata, is still capable of infecting newsubstrates at that concentration. Exposures of 24 hours at 5 ppm are sufficientto control all fungi tested in this study. It should be noted that malachite greenis extremely toxic to Saprolegnia parasitica, as this species is unable to infest newseeds when treated with as little as 1 ppm for one minute. This undoubtedly120 RICHARD MARTIN Vol. 68explains why malachite green is effective in controlling certain fungus infections,many of which are due to this particular fungus (Scott and O'Bier, 1962). Itmust be stressed that, if malachite green is to be used, the fungus causing theinfection should be identified to species so as to determine the nature of adequatetreatment. According to Arasaki et al. (1958), as little as 0.02 ppm of malachitegreen will restrain the growth of saprolegniaceous hyphae and sporangia. Scottand Warren (1964) reported that 2 ppm of malachite green, when applied to afungus for 24 hours, would stop all growth of Saprolegnia sp., Achlya americana,and Pythium sp. High concentrations, however, when applied for only shortperiods of time are not completely effective (table 1).O'Donnell (1941) recommends a treatment of 10 to 30 seconds in a 67-ppmsolution as being non-toxic to 18 species of fish and yet effective as a fungicide.This concentration, tested by the author on seven species of fungi for 20 seconds,was found to be toxic to all except A. ambisexualis (male), which did infest newseeds after four days. Quite possibly, therefore, other species of fungi may alsobe refractory to this treatment.When fish are exposed for longer periods, even to much lesser concentrations,malachite green proves highly toxic to the fish. Willford (1967) reports thatbluegills, and rainbow, brook, brown, and lake trout show 50% mortality whentreated with concentrations of 0.6 ppm or less at 12°C. At 17°C, the 24-hr LC5o(lethal concentration at which 50% of the test fish die) for channel catfish was0.21 ppm.It would appear that, if absolute control of parasitic fungi is to be obtainedwith long term (12-24 hr) treatments, concentrations that are lethal to the fishwould have to be used. If higher concentrations and shorter treatment durationsare used, the treatment may not be effective in controling all species of fungi.At best malachite green is an imperfect tool with limited value and should be usedonly where identification of the fungus species present shows that, at certainspecific concentrations and time intervals, the method would be effective.Some promising results in the control of parasitic fungi have already beenobtained with ozone (Benoit and Matlin, 1966), and another aniline dye, acrifiavine(Martin, 1967), though the latter must be cleared by the Food and Drug Adminis-tration before it can be used to treat fishes. Further investigations with this andother as yet untested compounds could, in the near future, make the use of mala-chite green obsolete.ACKNOWLEDGMENTSThe author wishes to acknowledge the assistance of Dr. John A. Schmitt,Department of Botany at The Ohio State University, who provided facilities, themajority of the experimental fungi, and reviewed this manuscript. Thanks aredue to Dr. L. S. Putnam, Department of Zoology at The Ohio State University,who provided substantial aid and assistance with all phases of this work. Theauthor also wishes to express his gratitude to J. Timothy Bowen, Fish HatcheryBiologist at the LaCrosse Fish Control Laboratory near LaCrosse, Wisconsin,and Dr. Glen L. Hoffman, Parasitologist with the Eastern Fish Disease Laboratory,Kearneysville, West Virginia, both of whom reviewed portions of the manuscriptand made useful suggestions.LITERATURE CITEDAgersborg, H. P. Kjershow. 1933. Salient problems in the artificial rearing of salmonid fisheswith special reference to intestinal fungisitosis and the cause of white-spot disease. Trans.of the Amer. Fisheries Soc, 63: 242.Arasaki, S. K., K. Nozawa, and M. Mizaki. 1958. On the pathogenicity of water mold. Bull.Jap. Fisheries Soc, 23: 593-598 as seen in Fishery Leaflet 564, Bureau of Sport Fisheriesand Wildlife.Benoit, R. F. and N. A. Matlin. 1966. Control of Saprolegnia on eggs of rainbow trout (Salmogairdneri) with ozone. Trans, of the Amer. Fisheries Soc, 95(4): 430-432.No. 2 FUNGICIDAL EFFECT OF MALACHITE GREEN 121Johnson, H. E., C. D. Adams, and R. J. McElrath. 1955. A new method of treating salmoneggs and fry with malachite green. Prog. Fish-Cult. 17(1): 76-78.O'Donnell, John. 1941. A new method of combating fungus infection. Prog. Fish-Cult.(56): 18-20.Martin, Richard L. 1967. A comparative investigation of the effect of various concentrationsof malachite green and of acriflavine on fungi associated with diseased fish. UnpublishedM.Sc. thesis, The Ohio State University. 000 p.Nigrelli, R. F. 1943. Causes of diseases and death of fishes in captivity. Zoologica, 28: 203-216.Scott, Wm., and A. H. O'Bier. 1962. Aquatic fungi associated with diseased fish and fisheggs. Prog. Fish-Cult. 24(1): 3-15.Scott, W. W., and C. O. Warren, Jr. 1964. Studies of the host range and chemical controlof fungi associated with diseased tropical fish. Va. Agric. Exper. Sta., Blacksburg, Va.Tech. Bull. 171.Vishniac, H. S., and R. F. Nigrelli. 1957. The ability of Saprolegniaceae to parasitize platyfish.Zoologica, 42(4): 131-134.Willford, W. A. 1967. Investigations in fish control. The toxicity of 22 therapeutic com-pounds to six fishes. Bureau of Sport Fisheries and Wildlife Resource Publication (In Press).

The Effect of Malchite Green as a Fungicide

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The Effect of Malchite Green as a Fungicide

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