Measurement of Malachite Green Distribution in Vertical Flow Incubator Trays during a Salmonid Egg Flush Treatment

Transcription

1 Measurement of Malachite Green Distribution in Vertical Flow Incubator Trays during a Salmonid Egg Flush Treatment J. O. Jensen 'and B. B. Windecker Department of Fisheries and Oceans Fisheries Research Branch Pacific Biological Station Nanaimo, British Columbia V9R 5K6 June 1982 Canadian Technical Report of Fisheries and Aquatic Sciences No Govemment of Canada Goo emernenl du Canada FlShenes and Oceans Pl!ches er'oceans

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3 Canadian Technical Report of Fisheries and Aquatic Sciences No. June 1982 MEASUREMENT OF MALACHITE GREEN DISTRIBUTION IN VERTICAL FLOW INCUBATOR TRAYS DURING A SALMONID EGG FLUSH TREATMENT by J. O. T. Jensen and B. B. Windecker Department of Fisheries and Oceans Fisheries Research Branch Pacific Biological Station Nanaimo, British Columbia V9R 5K6

4 - if - (c) Minister of Supply and Services Canada 1982 Cat. No. Fs 97-6/1106 ISSN

5 - iii - ABSTRACT Jensen, J. O. T., and B. B. Windecker Measurement of malachite qreen distribution in vertical flow incubator trays during a salmonid egq flush treatment. Can. Tech. Rep. Fish. Aquat. Sci. 1106: iii + 11 p. A technique for the colorimetric determinat ion of malachi te green is described. Two flush treatments were carried out with vertical flow incubators. Malachite green concentrations and retention times in each tray were found to vary depending on tray location and tray loading. The use of malachite green as a funqicide for salmonid eggs is discussed. Key words: malachite green, measurement, fungicide, salmonid eggs, flush treatment, exposure time. RrSUMt Jensen, J. O. T., and B. B. Windecker Measurement of malachite green dist r ibution in vert ical flow incubator trays during a salmonid egg flush treatment. Can. Tech. Rep. Fish. Aquat. Sci. 1106: iii + 11 p. Ce rapport presente une technique de determination colorimetrique du vert malachite. Deux rin9ages ont ete effectues dans les incubateurs a circulation verticale. Les concentrations et les periodes de retention du vert malachite dans chaque claie variaient selon la position et Ie chargement des claies. On discute de l'usage du vert malachite comme fonqicide pour les oeufs des salmonides. Mots-cles: vert malachi te, Quanti fication, fongicide, oeufs de salmonides, rin9age, temps d'exposition.

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7 INTRODUCTION Malachite green (C23H2SClN2)' 4-[p-(dimethylamino)-a-phenylbenzylidene]-2,S-cyclohexadien-l-ylidene dimethylammonium chloride, has been widely used as a fungicide in salmonid fish culture for many years. Its use continues despite recent evidence implicating it as a carcinogen (Nelson, 1974). Three methods have evolved for treating salmonid eggs against water-borne fungal infection (e.g. Saprolegnia). Two of these, the dip or bath method and the constant flow method, expose eggs to a known concentration of malachite green for a given period of time (Burrows, 1949; Johnson et a1., 19S5; Leitritz and Lewis, 1976; Wood, 1979). The third method, the flush treatment, is widely used owing to its simplicity. The technique consists of pouring a given volume of a concentrated solution of malachite green into the inflow end of the incubation trough, box, or vertical flow incubator stack. The 'slug' of fungicide then is flushed past the eggs over a short interval, usually about 10 min, depending on incubator dimensions, water flow rate, and egg loading density. Several flush treatment recommendations have been published (Table 1), differing in stock solution concentration and treatment frequency. These recommendations are based on empirical evidence of successful fungicidal effectiveness and subsequent normal development and survival of eggs. The flush method, however, does not expose all eggs to equal dosages of fungicide. Malachite green concentration and exposure time would vary, depending on the location of a batch of eggs relative to the inflow and outflow of the system. Some data are available on the relation between malachite green concentration and contact time required to control fungus (Olah and Farkas 1978). However, information on the effects of exposure of salmonid eggs to malachite green is limited and safe exposure levels are based primarily on empirical evidence (Nelson, 1974). The non-uniform applied dosages (concentration and exposure time) that occur with a flush treatment increase the possibility of under-exposure, resulting in incomplete fungicidal effectiveness, and over-exposure, resulting in possible injury to eggs or egg loss. To ensure against such possibilities, standard bioassays should be conducted to determine fungicidal efficacy and lethal thresholds for eggs with a range of stock solution concentrations, contact times and treatment frequencies. Before such tests can be carried out changes in malachite green concentration and exposure times must be determined for particular treatment conditions. A literature review (Nelson, 1974) indicated that a simple technique for malachite green measurement had not been developed. This report deals with (1) the development of a colorimetric technique for measuring malachite green concentration, and (2) measurement of malachite green concentration and retention times during a flush treatment with known water flow characteristics, those shown in the last entry of Table 1.

8 - 2 - METHODS AND MATERIALS Malachite green is water soluble and has a stable blue-green colour with an absorption maximum at 617 nm in a slightly acid medium ranging from ph 5.5 to 6.8 (Nelson, 1974). Therefore, to measure malachite green co1orimetrica11y the water samples should be buffered to yield a ph in this range. A phosphate buffer was prepared (12.5 g KH2P04 and 1.99 g K2HP04 dissolved in 1 L of distilled water) to yield a ph of Samples of malachite green standards then were buffered, volumetrically, at a ratio of 9:1 (samp1e:buffer). The ph of buffered samples was determined with a Fisher Accumet 320 ph meter. A 2-h period was found necessary for colour stabilization to occur after buffer addition. Absorbance measurements were obtained for standard concentrations of malachite green, ranging from 1 to 10 mg L-1, on a Bausch and Lomb Spectronic 70 spectrophotometer at 617 nm with a 1-cm path length. Two flush treatment tests were conducted using a stack of eight vertical flow incubation trays (Heath Techna Corp.). The first test was performed with empty trays with an available volume, when filled with water, of 12 L per tray. The second test was performed with trays half-filled with porcelain (Inta1ok) saddles, to simulate egg loading, with an equivalent available water volume of 10.7 L per tray. Water flow rate was set at 12 L min-1 and 20 m1 of a 20 g L-l stock solution (i.e. 400 mg malachite green) were added to the top tray at the point of water inflow. This represents a typical application of the flush treatment routinely used at a salmon hatchery in British Columbia, Canada (W. E. McLean, personal communication, Table 1). Commencing with the appearance of dye at the outflow of the top tray, 10 m1 samples were taken at 0.s-1.0-min intervals from the outflow trough of each tray. Samples with malachite green concentrations greater than 10 m1 L- 1 were diluted to provide final concentrations in the range of calibration. The samples were buffered and analyzed for malachite green concentration as described. RESULTS The ph of the buffered malachite green standards and samples remained constant at ph 6.3 from, at least, 2 to 48 h. The absorbance measurements of the standards (Table 2) were used to construct a calibration curve (Fig. 1). The least-squares regression equation for this relationship was Y= O.OlsX, where Y=absorbance and X=mg L-1 malachite green (r=0.9998). Malachite green concentrations from the flush treatment tests were plotted against time to illustrate the differences in dosage of malachite green (concentration x time) occurring in the individual trays.

9 - 3 - In the first test (Fig. 2) the top tray outflow reached a max1mum of 18.9 mg L-l 1.3 min after the stock solution was added, and dropped rapidly to 0.9 mg L-l after 3.3 min. In contrast, the bottom tray reached a peak concentration of 6.4 mg L-l after 6.8 min and a concentration of 1.1 mg.l-l still remained after 12.8 min. The second test (Fig. 3) with less tray volume available for water showed a similar trend, but higher and sharper concentration maxima occurred. The top tray reached a maximum concentration of 25.0 mg L-1 after 0.8 min and declined to 0.4 mg.l-1 after 3.3 min. The bottom tray concentration peaked at 8.0 mg L-l after 5.8 min and fell to 1.1 mg.l-l after 9.8 min. DISCUSSION The colorimetric method developed for quantitative measurement of an aqueous solution of malachite green appears reliable and accurate. The dilution water used in the tests was very soft; water hardness varying annually from mg.l-l as CaC03. Some modifications might be required 1n buffering hard water to maintain ph in the proper range. The concentration curves (Fig. 2, 3) for the two flush treatment tests illustrate the wide variety of exposure concentrations and durations that eggs experience depending upon their location in a vertical stack of trays. These differences will occur independent of rate of water flow through the vertical tray stack, of individual tray volume, or of egg loading rate. Furthermore, altering water flow rate, fungicidal stock concentration, and egg loading rate per tray will result in a new set of concentration curves for each tray. In a production hatchery with millions of eggs of various species and sizes, and where strict control of water flow rate is not possible, it is likely that each stack of incubator trays will have a unique set of concentration curves during a flush treatment. The chances of over-exposure and under-exposure to malachite green occurring therefore are increased when the flush method is used in a production hatchery. The concern regarding under-exposure to malachite green is that of incomplete fungicidal efficacy. Olah and Farkas (1978) found that a 5-min exposure to 3 mg.l-l of malachite green resulted in 100% mortality for one strain of Saprolegnia. At 2 mg.l-l, total mortality did not occur until after a 60-min exposure [effective time to 50% mortality (ET50) about 3 min]. At 1 mg L-l only about 75% mortality occurred after 24 h (ET50, about 35 min). Martin (1968) found a strain of~. parasitica to be even less tolerant to malachite green, a 5-min exposure period to 1 mg.l-1 being required to stop growth. On the other hand, a strain of S. feran was still capable of propagation when similarly treated, while exposure to 5 mg.l-l for 5 min stopped growth. The Achlya species of fungus tested by Martin (1968) was more resistant to malachite green; for example, growth of!. oblongata was not stopped until after a l-h exposure to 15 mg.l-l of malachite green.

10 - 4 - Fungi of the genus Saprolegnia frequently are encountered growing on fish eggs. Of 64 isolates obtained over two years from various locations in 14 American states, Scott and O'Brien (1962) found that 63% of the isolates identified consisted of Saprolegnia. At present there are not enough data to be definitive on the concentration-time aspects of exposure of Saprolegnia or other fungi to malachite green. Therefore, specific recommendations cannot be made. Similarly, there are not enough data on concentration-time aspects of exposure of salmon eggs to malachite green to allow recommendations on "safe" dosages to eggs. Therefore, as an interim recommendation, based largely on the data of Olah and Farkas (1978), we suggest a minimum exposure time of 5 min at 3 mg.l-l malachite green for treatment of salmonid eggs. This should result in 100% mort ali ty of Saprolegnia, the more frequently occurring fungus infecting incubating salmon1d eggs. The basic recommended dosage of 3 mg.l-l for 5 min was not achieved in any of the trays during the flush treatment tests with simulated egg loading (Fig. 3). The exposure times for concentrations ~ 3 mg.l-l ranged from 1.9 min in the top tray to 3.8 min in the bottom tray. Therefore, i.t is possible for the given flow rate, tray volume, and stock concentration that this treatment is not 100% effective. In a flush treatment it also is obvious that contact times providing 100% mortality of Saprolegnia during peak concentrations (>3mg.L-l) may be less than the 5-min exposure recommended for 3 mg.l-l malachite green. We believe the recommended dosage (3 mg.l-l, 5 min) is reasonable until further evidence is available on the dosages required (concentration x contact time) to kill Saprolegnia without harming incubating eggs. Another fact to consider is that eggs may adsorb malachite green during a flush treatment, thereby reducing the effecttveness of the fungicide in the lower trays. Interim alternative flush treatment procedures, pending availabi lity of further data, are (1) to increase stock concentrat ion strength unt il a concentration of > 3 mg.l-l is maintained in each tray for at least 5 min or (2) to decrease flow rate (e.g, to 6 L.min-l ) so that retention time of malachite green would be increased in each tray. The first alternative would increase the danger of over-exposure to the eggs while the latter would requ1re a major, and often difficult, flow adjustment. The non-uniform fungicide concentrations and contact times that have been demonstrated for a flush treatment would tend to increase the danger of both under-exposure, result ing in incomplete fungus mort ali ty, and over-exposure, increasing the possibility of mortality or other sublethal effects in developing salmonid embryos. Thus, if flush treatments with malachite green are to be continued in salmonid hatcheries, we suggest that toxicity tests be conducted for both fungus and eggs, to establish optimum fungicidal concentrations in conjunction with safe limits for developing eggs. The quality of fry obtained from eggs used tn such trials should then be monitored, at least up to complete yolk absorption, to ensure that any delayed effects attributable to malachite green exposure are not overlooked.

11 - 5 - REFERENCES Burrows, R. E Prophylact ic treatment for control of fungus (Saprolegnia parasitica) on salmon eggs. Prog. Fish.-Cult. 11(2): Foster, F. J., and L. Woodbury. fungicide and anteseptic The use of malachite green as a fish Prog. Fish.-Cult. 18: 7-9. Johnson, H. E., C. D. Adams, and R. J. McElrath treating salmon eggs and fry with malachite green. 17(2): A new method of Prog. Fish.-Cult. Leitritz, E., and R. E. Lewis Trout and salmon culture (Hatchery methods). Calif. Dept. Fish and Game, Fish. Bull. 164: Martin, R. L Comparison of effects of concentrations of malachite green and acriflavine on fungi associated with diseased fish. Prog. Fish.-Cult. 30(3): Mazuranich, J. J., and W. E. Nielson White-spot disease of salmon fry. Prog. Fish.-Cult. 21(4): Nelson, N. C A review of the literature on the use of malachite green in fisheries. U.S. Bureau of Sport Fisheries and Wildlife, Western Fish Disease Laboratory, Seattle, Washington O'Donnel, D. J The disinfection and maintenance of trout hatcheries for the control of disease, with special reference to furunculosis. Trans. Amer. Fish. Soc. 74: Olah, J. and J. Farkas Effect of temperature, ph, Antibiotics, formalin and malachite green on the growth and survival of Saprolegnia and Achlya parasitic on fish. Aquaculture, 13: Robertson, R Malachite green used to prevent fungus on lake trout eggs. Prog. Fish.-Cult. 16(1): 38. Scott, W. W., and A. H. O'Brien. diseased fish and fish eggs Aquatic fungi associated with Prog. Fish.-Cult. 24(1): Wood, J. W Diseases of Pacific salmon, their prevention and treatment. Wash. Dept. of Fish. 2nd Edition.

12 Table 1. Summary of malachite green flush treatment recommendations. Stock solution Quant ity of Malach ite green Flow rate Treatment Freque ncy Stock added Source g'l -1 (ml) (mg) (L'min-1) Foster and normal? Every day or two Woodbury (1936 ) O'Donald (1944 ) top 31.0? Every second day or 29.3 middle 15.5 daily Robertson (1954) Twice daily Double dos age if silt present Wood (1974) R When necessary (j\ Leitritz and R Every second day or Lewis (1976) daily if req uired McLean (1979 ) Twice week ly (Personal communication)

13 - 7 - Table 2. Absorbance readings of buffered malachite green standards. Malachite green concentration (mg L- 1 ) 1 Absorbance readingsa ai-em pathlength at 617 nm.

14 - 8 - Fig. 1. (ph=6.3) by least X=mg L-1 Malachite green calibration curv~ for buffered samples measur~d at ~=617 nm with a l-cm path Length. Line fitted squar~s regression equation Y= SX (Y=absorbance, malachite green).

15 - 9 - w u z <t ~ (/) m <t / / // MALACHITE GREEN (mg.{-i)

16 Fig. 2. Malachite green concentrations during a flush treatment through 8 vertical flow incubation trays. Water volume per tray, 12 L; water flow rate, 12 L'min- l ; stock solution (20 g'l-l) added, 20 mi. Fig. 3. Malachite green concentrations during a flush treatment through 8 vertical flow incubation trays, half filled with Intalok Saddles. Water volume per tray, 10.7 L; water flow rate, 12 L min-i; stock solution (20 g.l-l) added, 20 mi.

17 ~ ;- ~ do E z w a:: C> w ~ ::r: u <l.-oj <l :E TRAY NUMBER SYMBOL TOP I t:: BOTTOM 8 '\l 0 5 O~L-~ ~.~~ ~~~~~:D~=-~~ ~ ~, o J II TIME (MIN.) , '"'Y do E z w a:: C> w ~ ::r: u <l.-oj <l ~ 20 IW\Y NUMHIH ~JYMBOL TOP I t::. 15 j BOTTOM 8 \l 10 5 O+---~--~---r-=~--~--~--~~~r-~r-~~--~~~==~ ~ o II TIME (MINJ