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Effects of Potassium Permanganate on the Sailfin Molly, Poecilia Latipinna, at Varying Salinity Levels

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PAGE 1

EFFECTS OF POTASSIUM PERMANGANATE ON THE SAILFIN MOLLY, Poecilia latippinna, AT VARYING SALINITY LEVELS By EMILY N. MARECAUX A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2006

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Copyright 2006 by Emily N. Marecaux

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To my family and friends who have s upported me throughout my college career.

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iv ACKNOWLEDGMENTS My committee members, Ruth Francis-Floyd, B. Denise Petty, Scott P. Terrell, Kathleen H. Hartman, and Roy P. E. Yanong, have provided significan t contributions to this project. Jeff Hill provided extremely impor tant help with the statistical analysis of this project. Sherry Giardina provided he lpful guidance through the graduate student process. Scott Graves of the University of Florida Tropical Aquaculture Laboratory helped me set up the aquaculture tank system used for this pr oject. Jamie Holloway from the Department of Fisheries and Aquatic Sciences helped with the pilot study and gathering of supplies for this project. Patricia Lewis and Don Samuelson from the University of Florida College of Veterinary Medicine allowed for the use of the histology laboratory which was an integr al part of my project. R obert Leonard, Tina Crosby, Jen Matysczak helped gather behavioral observati on data. Chris Langeneck took the pictures of the sailfin mollies. Finally, the generous support from Segrest Farms was very much appreciated and the project could not have been completed without their donation. Thank you to everyone that helped this project come to completion.

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v TABLE OF CONTENTS page ACKNOWLEDGMENTS.................................................................................................iv LIST OF TABLES............................................................................................................vii LIST OF FIGURES.........................................................................................................viii ABSTRACT....................................................................................................................... ix CHAPTER 1 LITERATURE REVIEW.............................................................................................1 Introduction................................................................................................................... 1 The Sailfin Molly – Poecilia latipinna .........................................................................3 Potassium Permanganate – KMnO4..............................................................................5 Tables and Figures........................................................................................................9 2 PILOT STUDY...........................................................................................................11 Experimental Design..................................................................................................11 Results........................................................................................................................ .13 Discussion...................................................................................................................14 3 MATERIALS AND METHODS...............................................................................15 Experimental Procedures............................................................................................15 Experimental Fish and Quarantine Procedure.....................................................15 Acclimation Procedure........................................................................................16 Response Variables.............................................................................................17 Experimental Design..................................................................................................19 KMnO4 Treatment...............................................................................................19 Experimental Procedure......................................................................................20 Statistics..................................................................................................................... .21 4 RESULTS...................................................................................................................22 Water Quality..............................................................................................................22 Behavior......................................................................................................................2 2

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vi Histology.....................................................................................................................2 4 Mortality.....................................................................................................................2 7 5 DISCUSSION AND CONCLUSIONS......................................................................31 APPENDIX A PROCESSING SCHEDULE FOR THE SHANDON EXCELSIOR AUTOMATIC TISSUE PROCESSOR......................................................................39 B TYPICAL COMPOSITION OF INSTANT OCEAN SALT.....................................40 LIST OF REFERENCES...................................................................................................41 BIOGRAPHICAL SKETCH.............................................................................................45

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vii LIST OF TABLES Table page 1-1 The 96h LC50 levels for potassium permanganate (KMnO4) determined for juvenile and larval striped bass, Morone saxatilis ...................................................10 1-2 Dosages and target pathogens of KMnO4 based on literature review......................10 3-1 Experimental treatment combinations of KMnO4 concentrations and salinity levels......................................................................................................................... 19 3-2 Calculated KMnO4 demand for each salinity level..................................................20 4-1 Select water chemistry values for ex perimental tanks (n = 12) prior to KMnO4 treatments.................................................................................................................22 4-2 Kruskal-Wallis analysis of six-hour be havior scores by treatment combinations (salinity – KMnO4 concentration) (N = 3 tanks per treatment)................................23 4-3 Dunn’s multiple comparison test for be havior scores shows significant treatment groups.......................................................................................................................24 4-4 Kruskal-Wallis analysis of the slide score by the salinity/KMnO4 treatment combinations............................................................................................................25 4-5 Dunn’s multiple comparison test for hi stology slide scores shows significantly different treatments..................................................................................................25 4-6 Two-way ANOVA of the total percen tage mortality by the salinity and the KMnO4 concentration..............................................................................................28 4-7 One-way ANOVA analysis of the total mortality percentage by the treatment combinations............................................................................................................28 A-1 Processing schedule for the shandon ex celsior automatic tissue processor.............39 B-1 Typical composition of instant ocean salt................................................................40

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viii LIST OF FIGURES Figure page 1-1 The sailfin molly........................................................................................................9 4-1 Gills from sailfin mollies in 2 g/L salinity water treated with KMnO4....................26 4-2 Gills from sailfin mollies in 15 g/L salinity water treated with KMnO4..................26 4-3 Gills from sailfin mollies in 30 g/L salinity water treated with KMnO4..................27 4-4 Cumulative Total Percentage Mortality Over 7 days by Salinity (fresh 2 g/L, brackish 15 g/L, and salt 30 g/L) an d KMnO4 Concentration (0.0, 0.5, 1.0, and 3.0 mg/L)..................................................................................................................29 4-5 The cumulative total percentage mo rtality shown over time for the 2 g/L treatments.................................................................................................................29 4-6 Cumulative total percentage mort ality shown over time for the 15 g/L treatments.................................................................................................................30 4-7 Cumulative total percentage mort ality shown over time for the 30 g/L treatments.................................................................................................................30

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ix Abstract of Thesis Presen ted to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science EFFECTS OF POTASSIUM PERMANGANATE ON THE SAILFIN MOLLY, Poecilia latipinna, AT VARYING SALINITY LEVELS By Emily N. Marecaux May 2006 Chair: Ruth Francis-Floyd Major Department: Fisherie s and Aquatic Sciences Potassium permanganate (KMnO4) is used in fish culture for disease treatment, water clarification, rotenone detoxification, and historically for management of oxygen depletion. Most commonly, KMnO4 is used in freshwater systems at 2 mg/L to control ectoparasites, bacteria, and f ungi. Effective concentrations are determined by the KMnO4 demand of the water being trea ted. Although safe use of KMnO4 in freshwater systems is well documented, its toxicity to fish in saltwater systems is less well known. The sailfin molly, Poecilia latipinna a euryhaline species, was used as a model to test the toxicity of KMnO4 at varying concentrations and at different salinity levels. Target KMnO4 concentrations of 0.0, 0.5, 1.0, and 3.0 mg/L plus the KMnO4 demand were tested. Toxicity was te sted at salinity levels of 2, 15, and 30 g/L. Mortality rates and fish behavior were monitored throughout the experiment and tissue samples for histological analysis were taken at time zero, immediately post-treatment (12 hours), and at the end of the monitoring period (7 days).

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x The mortality rate was significantly highe r in the 30 g/L salinity, 3.0 mg/L KMnO4 treatment group than in any other treatmen t group (p < 0.001). The 15 g/L salinity, 3.0 mg/L KMnO4 treatment group was also found to be significantly different from the 15 g/L salinity 0.0 and 0.5 mg/L KMnO4 treatment groups ( p < 0.001). The 2 g/L salinity, 3.0 mg/L KMnO4 treatment was not found to be signi ficantly different. Dunn’s multiple comparison test indicated that treatments of 2 g/L, 15 g/L, and 30 g/L salinities treated with 3.0 mg/L showed significant changes in behavior resulting in the loss of equilibrium. Dunn’s multiple comparison test also indicated that treatments 15 and 30 g/L salinity, 3.0 mg/L KMnO4 concentration showed significa ntly different gill damage as indicated by secondary lamellar fusion, lifti ng of epithelial cell lin ing by expansion of the lamellar interstitium by inflammation and edema, and necrosis. Results from this study suggest that KMnO4 at concentrations of 0.5 and 1.0 mg/L may be safe for use in water containing sailfin mollies in water of salinities of 2, 15, and 30 g/L. However, KMnO4 should not be used at concentrations of 3.0 mg/L in 2, 15 or 30 g/L salinity water on the sailfin molly until further research is conducted. Toxicity of potassium permanganate increased in the higher salinity groups (15 and 30 g/L) compared to the low salinity group (2 g/L).

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1 CHAPTER 1 LITERATURE REVIEW Introduction Potassium permanganate, KMnO4, is an oxidizing agent that is used in fish culture for disease treatment, water clarification, ro tenone detoxification, and historically for management of oxygen depletion in fish ponds. Potassium permanganate has been used to treat external pathogens including fungus bacteria, and some parasites (Lay 1971; Masser and Jenson 1991; Noga 1996; Fran cis-Floyd and Klinger 1997; Plumb 1999; Carpenter et al. 2001; Stoskopf 1993; Bishop 2001; Straus and Gr iffin 2002; ThomasJinu and Goodwin 2004). KMnO4 can also be used for wate r clarification purposes (Lay 1971) by oxidizing organic material in the wa ter, forming precipitates that can be removed from the water by a filter. When ro tenone is used in water treatments, KMnO4 can be used as a counter agent to de toxify the water (Lawrence 1956). KMnO4 was also used to add oxygen to aquaculture ponds in si tuations of low dissolved oxygen levels in water (Lay 1971). This method is no longer practiced as KMnO4 did not raise oxygen levels significantly and may cause oxygen levels to decrease by killing the oxygen producing algae in the pond (Tucker and Boyd 1977). Many different treatment regimes have been developed over the years for the different uses of KMnO4 in aquaculture. However, use of KMnO4 in salt water is less common than in freshwater systems so less is known about what happens when it is used as a treatment in the marine environment. There is debate over the use of KMnO4 for

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2 treatment of marine fish and concern re garding the toxicity threshold to KMnO4 in marine species held in saltwater. Marking and Bills (1975) found that KMnO4 was more toxic to fish in waters with pH between 8.5 and 9.5 and total hardness of 300 mg/L as CaCO3 (compared to water with a total hardness of 12 mg/L as CaCO3). Stuart (1983) and Noga (1996) have suggested that KMnO4 is toxic to fish in saltwater because of the higher pH typically associated with saltwater, which may cause manganese dioxide to precipitate onto the gills. Natural seawater typi cally has a pH between 7.8 a nd 8.2 (Spotte 1992), which may be higher than the pH of so me freshwater systems, whic h are commonly between 6.8 and 7.2. In a freshwater efficacy study using KMnO4 at a dosage of up to 1.5 mg/L to treat ichthyophthiriasis in channel catfish, Ictalurus punctatus the pH was maintained at 8.5 +/0.1 (Straus and Griffin 2002), and the auth ors reported that no fish died in the effective treatment group (1.25 mg/L). This sugg ests that pH levels above those typically found in freshwater may not be the sole cause of KMnO4 toxicity to fish. Reardon and Harrell (1994) determined the KMnO4 concentration that caused 50% mortality of exposed population over 96 hours, 96h LC50, at different salinity levels up to 15 g/L for juvenile and larval striped bass, Morone saxatilis In this study juveniles were most tolerant of KMnO4 at a salinity level of 5 g/L, wh ile larvae were most tolerant of KMnO4 at a salinity level of 3 g/L. Both j uvenile and larval striped bass were least tolerant of KMnO4 at salinity levels of zero. The 96h LC50 levels that Reardon and Harrell (1994) reported are depicted in Table 1-1 (see page 10). There was a significant decrease in the 96h LC50 level at salinity levels of 0, 10, and 15 g/L compared to the 5 g/L treatment for juveniles with KMnO4 being most toxic

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3 to juveniles at the 0 g/L salinity followed by th e 15 g/L salinity. It was suggested that the greatest toxicity occurred in 0 g/L water becau se this was the salinity where the greatest osmotic imbalance occurred. This experiment showed that margin of animal safety may narrow when KMnO4 is applied at higher salinities, but it did not demonstrate if the trend continued as salinity incr eased above 15 g/L. The Sailfin Molly – Poecilia latipinna The sailfin molly, Poecilia latipinna, formerly described and named Mollienesia latipinna by Charles Alexandre Lesueur in 1821 (R obins 2003), is from the family Poeciliidae, comprising over 190 species (Paren ti and Rauchenberger 1989). The natural distribution of the sailf in molly is fresh, brackish, and sa lt waters of Florida, Mexico, Texas, South and North Carolina, and Vi rginia (Petrovicky 1988; Robins 2003; Courtenay and Meffe 1989). Non-indigenous po pulations are established in the western U.S. (Arizona, California, and Nevada), Ha waii, Canada, Central America, Singapore, Australia, New Zealand, Guam, and the Ph ilippines (Courtenay and Meffe 1989). The sailfin molly prefers lowland areas such as marshes, lowland streams, swamps, and estuaries (Robins 2003). The sailfin molly is a fusiform shaped small fish (15-53 mm total length) with a small head and upturned mouth (Robins 2003)(see Figure 1-1, page 9). The dorsal fin is greatly enlarged in mature males compared to those of mature females. The dorsal fin is used as a display to attract females for re production. Only dominant males display the dorsal fin. Subordinate males use the “ambus h” technique for breed ing (Balsano et al. 1989). The “ambush” technique refers to th e chance that the dominant male becomes distracted, allowing the subordina te male to breed with the female before being chased away. Males have a modified anal fin cal led the gonopodium, which is used for internal

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4 fertilization. At rest the gonopodium points caudally, but during repr oduction, the gonopodium is pointed forward and is inserted into the female in a quick motion, which results in sperm being deposited into the female. The female molly can store the sperm deposited by the male. The gestation period is 3-4 weeks. Females are viviparous and give birth multiple times during the year (Robins 2003). The sailfin molly, Poecilia latipinna is a euryhaline species that can tolerate salinities as high as 70 to 80 g/L (Nordlie et al. 1992). Gustafson (1981) used short-term salinity acclimation as a method to evalua te the influence of salinity on plasma osmoregulation and routine oxygen consumption. Gustafson (1981) alte red the salinity at a rate of 4.14 g/L per day for sailfin mollies of brackish water origin (mostly 10-20 g/L, but ranged from 4-35 g/L) and for sailfin mollies of freshwater origin (no information on the salinity level given). Frank Nordlie (The University of Florida, personal communication) recommended salinity level adju stments at a rate of 5 g/L every five days for proper osmoregulation balance. Sa ilfin mollies are highly adaptable to changing salinity ranges that are found in their natural habitat. For aquaculture purposes, salinity up to 3 g/L is considered freshwater (Chapman, The University of Florida, personal comm unication), while brackish water ranges from 3.0 g/L to 29 g/L and saltwater is 30 g/L and above. Some species are more sensitive to salinity than others, ther efore, it is necessary to know the limits of the species being used for experimentation. The sailfin molly is primarily an herbivor e, eating plants and algae, but is also opportunistic and will eat other food items including detritus or insect larvae and cannibalism has been reported (Meffe and Snelson 1989). The sailfin molly is a prey

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5 item for many predators. Predators that eat this fish include reptiles, birds, other fishes, amphibians, and insects. The sailfin molly is also popular in the a quarium trade and is available in a wide variety of colors through domestication. It has also been used for research and for biological control of mosquitoes (Courtenay and Meffe 1989). Potassium Permanganate – KMnO4 Historically, the Environmental Protec tion Agency (EPA) (1985), registered KMnO4 for use in cooling towers, evaporative co ndensers, air wash systems, ornamental ponds, cooling fountains, aquaria, human dri nking water, poultry drinking water, for surface disinfection, sanitization, and as a deodorizer. However, KMnO4 is now exempt from registration by the EPA because it does not pose unreasonable risks to public health or the environment. Currently KMnO4 is a U.S. Food and Drug Admini stration (FDA), investigational new animal drug (INAD) under investigati on by Carus Chemical Company (Peru, IL) and Stuttgart National Aquaculture Research Center (SNARC) (Stuttgart, AR). FDA approval will allow for the legal use of KMnO4 in water containing food fish. To gain approval from the FDA, Carus Chemical Comp any is responsible for research on product chemistry and mammalian toxicology, while SNARC is responsible for research on efficacy, human food safety, target animal sa fety, and environmental safety (Straus, SNARC, personal communication) Regulatory action on KMnO4 has been deferred pending the outcome of current research. As a therapeutant for fish, KMnO4 has been used as an external bactericidal and fungicidal agent. Formulations of KMnO4 are available as ready-to -use liquids, pellets or tablets, powder, or crystals. The active ingred ient is the permanganate ion. It functions as a strong oxidizing agent that is corrosive and burns any organic material it comes into

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6 contact with. For this reason it is effective if used in fish culture to control external disease-causing agents including bacteria, f ungi, and some parasites. The oxidizing activity is also the primar y problem for treated fish. As an oxidizing agent, KMnO4 is able to add oxygen, remove hydrogen, or remove electrons from an element or compound (Carus Chemical Company 2004). For example in drinking water treatments, KMnO4 is able to oxidize soluble manganese and iron into manganese dioxide and iron oxide, which are insoluble and can be removed by filtration. The effectiveness of KMnO4 is related to the amount of oxidizable material in the water, i.e. organic material and other elements (inorganic) that may be oxidized. This is referred to as the KMnO4 demand of the water (Tucker 1984). Engstrom-Heg (1971) developed a test, using a spectropho tometer, to determine the KMnO4 demand of the water to be treated. Later, Boyd (1979) deve loped a quick visual test to determine the KMnO4 demand of the water to be treated. He treated 1,000 mL samples of water with 0, 1, 2, 3, 4, 6, 8, 10, and 12 mg/L of KMnO4 in separate containers; after fifteen minutes, the container with the lowest concentration that was still pink was considered the KMnO4 demand of the water. Potassium permanganate is rendered in active by organic material (Tucker and Boyd 1977), therefore, in a recirculating syst em the biological filter may be affected because organic material, including the nitrifyi ng bacteria, may be oxidized. However, it is unclear if KMnO4 has a significant effect on the e fficiency of the biofilter (Spotte 1992). One study (Levine and Meade 1976) de monstrated that a treatment of KMnO4 inhibited nitrification 86%, while another study (Collins et al. 1975) demonstrated that

PAGE 17

7 KMnO4 had no effect on nitrification. If the f ilter has a build-up of organic material, it will increase the KMnO4 demand of the water if the filter is left online during treatment and the biological portion of the filter may be damaged. Tucker (1989) developed a method to estimate the required treatment of KMnO4 based on the 15-minute KMnO4 demand of the water. The calculation from his work is 2.5 multiplied by the value obtained in the fift een-minute test. However, the level of KMnO4 needed to control an ichthyophthirias is outbreak in an efficacy study was 1.25 mg/L (Straus and Griffin 2002). In that st udy it was determined that using Tucker’s method, the treatment rate indicated would be 1.0 mg/L. This implies that Tucker’s method is not a “fail-safe” method for determining treatment dosages. Another recommendation for compensation of the KMnO4 demand is to add 2 mg/L to the KMnO4 demand of the water (Plumb 1999). Other treatment recommendations found in the literature are summarized in Table 1-2 (see page 10). The toxicity margin of KMnO4 is narrrow (1-3 mg/L) (Plumb 1999). Toxicity levels have been determined for carp fry as an LC50 ranging from 37.5 to 48 mg/L at 26C and 45 to 37.5 mg/L at 32C, at 24 and 48 hours, respectively (Ghosh and Pal 1969). The pH was maintained between 7.8 and 8.2. Studies have shown that 20 mg/L KMnO4 is toxic to guppies and 3.2 mg/L KMnO4 is toxic to catfish (Scott and Warren as cited by Lay 1981), however KMnO4 demand or exposure time is not reported. Lawrence (1956) reported that toxicity levels of KMnO4 were 3 mg/L for bluegills, 4 mg/L for largemouth bass, and 6 mg/L for goldfish. Finally Lawrence (1956) showed a toxicity level of 5 mg/L KMnO4 for fathead minnows at a temperature of 68F in a prolonged bath (no method for removing the KMnO4 was employed), but restocking the

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8 aquaria 24 hours after treatment showed that the water was no longer toxic to fish at that time. Toxicity of KMnO4 decreases with increasing KMnO4 demand (Tucker and Boyd 1977). Potassium permanganate tolerance is dependent on water quality, exposure time, and the species of fish. Potassium permanganate efficacy studies have been conducted with columnaris and ichthyophthiriasis. Straus and Griffin (2002) reported th at a treatment of 1.25 mg/L KMnO4 was effective against ichthyophthirias is, meaning that no trophonts were found on treated channel catfish, Ictalurus punctatus In that study, treatments were administered daily after a complete water ch ange. The pH was maintained at 8.5 +/0.1 with a temperature of 17C +/2.5 and the study was run until the control fish died. In a challenge against columnaris, KMnO4 applied at a dose of 2 mg/L as an indefinite bath at a temperature of 22C, reduced mortality to 69% compared to the 100% mortality in infected control channel catfish, Ictalurus punctatus (Thomas-Jinu and Goodwin 2004). A fish that has been treated with KMnO4 may show signs of tissue damage depending on the dose of KMnO4. Darwish et al. (2002) fo und that chan nel catfish, Ictalurus punctatus treated with KMnO4 (0.438, 1.315, and 2.190 mg/L) at a pH of 7 +/0.2, showed microscopic lesions in the gill s using a routine hematoxylin and eosin histology stain, while liver and trunk kidney showed no lesions. The fish treated with 0.438 mg/L KMnO4 showed mild hypertrophy and spongios is of the epithelium of the gill filaments and lamellae. The fish treated with 1.315 and 2.190 mg/L KMnO4 showed extensive gill epithelial hype rplasia, lamellar fusion, and obl iteration of the interlamellar spaces with inflammatory exudates cont aining necrotic epithelial cells.

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9 Potassium permanganate is a strong oxidiz er and contact with other materials may cause fire; however by itself it is stable and wi ll keep indefinitely if stored correctly. Potassium permanganate is incompatible with the following substances: formaldehyde, powdered metals, alcohol, arsenites, brom ides, iodides, phosphorous, sulfuric acid, organic compounds, sulfur, activ ated carbon, hydrides, strong hydrogen peroxide, ferrous or mercurous salts, hypophosphites, hyposulfites, sulfites, peroxides, and oxalates. Care should also be taken when handling potassi um permanganate; it is corrosive and may cause burns to any area of contact and ma y be harmful if swallowed or inhaled. (Mallinckrodt Baker, Inc. 2001) Potassium permanganate is an important chemical in aquaculture because of its availability and its various uses. The goa l of this study is to determine if KMnO4 creates toxicity problems for sailfin mollies held in saltwater compared to cohorts maintained in freshwater. This study will examine the effect of KMnO4 on the sailfin molly maintained at different salinity levels and at different KMnO4 concentrations. Tables and Figures A B Figure 1-1. The sailfin molly. A. Th e gonopodium on this male sailfin molly is indicated by the arrow. B. A group of sailfin mollies. Photo credit: Chris Langeneck.

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10 Table 1-1. The 96h LC50 levels for potassium permanganate (KMnO4) determined for juvenile and larval striped bass, Morone saxatilis Juveniles (One-month old) Larvae (18-day old) Salinity Level g/L 96h LC50 Level mg/L KMnO4 Salinity Level g/L 96h LC50 Level mg/L KMnO4 0 0.96a 0 1.02a 5 3.26b 3 2.11b 10 1.63c 6 1.41ab 15 1.48c 9 1.73ab *Data taken from Reardon and Harrell (1994). Means with identical superscript are not significantly different at the 5% level using the SNK test. Table 1-2. Dosages and target pathogens of KMnO4 based on literature review. Recommended dose of KMnO4 (mg/L) Duration of treatment Treatment Use References 1000 10-40 second bath fungi, protozoa Carpenter et al. 2001; Noga 1996; Lay 1971 100 5-10 minute bath fish lice Carpenter et al. 2001; Noga 1996 20 1 hour crustaceans, protozoa Stoskopf 1993 10 30 minutes fungi, protozoa Lay 1971 5 30-60 minute bath ectoparasites, gill/skin bacterial infections Carpenter et al. 2001; Noga 1996 2 prolonged immersion or bath, indefinite, at least 4 hours, or at least 12 hours ectoparasites, gill/skin bacterial infections, protozoa, oxidation/ detoxification of hydrogen sulfide Bishop 2001; Noga 1996; Thomas-Jinu and Goodwin 2004; FrancisFloyd and Klinger 1997; Plumb 1999; Masser and Jensen 1991 2-5 indefinite crustaceans, protozoa Stoskopf 1993 2 prolonged bath counteract rotenone Lawrence 1956 2.5 flush for 4 consecutive days bacterial gill disease in salmonids Stoskopf 1993 2 flush ectoparasites, gill/skin bacterial infections Noga 1996

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11 CHAPTER 2 PILOT STUDY Experimental Design A pilot study was conducted at the Univers ity of Florida Department of Fisheries and Aquatic Sciences wet lab (Gaine sville, Florida) to determine KMnO4 treatment levels to be tested. Six, 37.85L tanks (filled to 30 L) each with one airst one, were set up at a salinity level of 5 g/L. Salinity was tested using a refractometer (Aquatic Eco-Systems, Apopka, Florida). Thirty-six sailfin mollies, Poecilia latipinna (mean weight = 2.3 grams) were received from Segrest Farm s (Gibsonton, Florida) and six fish were arbitrarily placed into each tank. Fish were fed once daily (1% of total biomass of tank) a flake fish food (Zeigler Tri-color flake, Ga rdners, Pennsylvania). Fifty percent water changes were conducted every other day in conjunction with salin ity adjustments. Saltwater was made with Instant Ocean salt mix (Aquarium Systems, Mentor, Ohio). Salinity was adjusted so that two tanks each were established at 2 g/L, 15 g/L, and 30 g/L, respectively, over a time pe riod of fourteen days. In an effort to meet a deadline, the salinity level was altered 5 g/L every othe r day until the desired salinity was reached, with the exception of the 2 g/L tanks, which we re adjusted by an incr ement of 3 g/L. The salinity level of 2 g/L was chos en over 0 g/L because captive sa ilfin mollies prefer to live in water that contains some salt (Petty, Univ ersity of Florida, pe rsonal communication). The treatment level of KMnO4 to be tested was 2 mg/L and 6 mg/L; one tank of each concentration (2 and 6 mg/L) was represente d at each salinity (2, 15, and 30 g/L).

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12 The KMnO4 demand of the water was determined to be 0.20 mg/L KMnO4 for 30 g/L salinity water, 0.15 mg/L KMnO4 for 15 g/L salinity water, and 0.10 mg/L KMnO4 for 2 g/L salinity water. Actual concentrations of KMnO4 were not determined because chloride interferes with typical spectrophotometric tech niques that might be used (Delfino, The University of Florida, 2004) because it reacts with KMnO4. Each salinity would require its own standa rdization curve and the highe r salinity water would produce a less reliable curve because th e active concentration of KMnO4 would be changing with increased contact time (some of the i norganic compounds, incl uding chloride, of saltwater reduce KMnO4). The 16th day after fish arrival a fifty pe rcent water change was done on each tank and one fish was sampled from each tank for histologic assessment of tissue prior to KMnO4 exposure. The designated KMnO4 treatment (either 2 or 6 mg/L KMnO4) plus the KMnO4 demand was added to the tanks and beha vior and mortality was monitored for twelve hours. Behavioral observations were re corded to aid in the design of the scoring system for the larger experiment at time zero (0), 0.08, 0.16, 0.33, 0.66, 1.5, 3, 6, and 12 hours. Each tank was dosed with KMnO4 and then fish were observed for behavioral alterations for 2 minutes until all tanks had b een observed at this observation time. This required two people to physica lly monitor the tanks for the first four time periods (zero, 0.08, 0.16 and 0.33 hours). All fish were sampled fo r histological analys is either at the time of their death (mortalities were mon itored hourly) or following twelve hours KMnO4 exposure. Fish that survived the treatme nt were euthanized with buffered (sodium bicarbonate) tricaine methanesulfonate (1 g/L dose) (Finquel, Ar gent Laboratories, Redmond, WA) before fixation. Whole fish were fixed in 10% neutral buffered formalin.

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13 After 48 hours of fixation whole fish were separated into two hi stology cassettes; one cassette contained complete, whole gill sections and the other cassette contained three sections of body tissue. After the gills we re removed the sample was cut into three transverse sections (or steaks) – cuts were made at the posterior eye, just cranially to the pectoral fin and at the anus. Tissue was pr ocessed according to the schedule listed in Appendix A. Results One hundred percent mortality was observed in all tanks at all salinities (2, 15, and 30 g/L) treated with 6 mg/L KMnO4. All fish from the 30 g/L salinity water treated with 6.0 mg/L KMnO4 died by the third hour of obser vation, while the 2 g/L and 15 g/L salinity group fish treated with 6.0 mg/L KMnO4 all died by the sixth hour of observation. Mortality was not observed in the three other tanks treated with 2 mg/L KMnO4. Behavior observations were recorded an d it was determined that fish generally exhibited the following behavi ors in sequential/chronological progression 1) increased opercular movement; 2) erratic swimming; 3) intermittent loss of equilibrium or lying on the bottom resting; and 4) the complete loss of equilibrium. Microscopic examination of control tissue samples were within normal lim its for the gills and other internal organs (e.g. kidney, liver, spleen, intestine). All gill tissue samples collected from KMnO4 treated fish showed mucous cell infiltra tion and hyperplasia and inflammatory cell infiltration and hyperplasia. The 15 g/L a nd 30 g/L salinity treatment at 6.0 mg/L KMnO4 showed severe inflammation, expansi on of the lamellar in terstitium by edema and inflammatory cells with lifting off of th e epithelium, lamellar fu sion and necrosis of the gill epithelium. The gill samples of fish from the 30 g/L salinity, 2 mg/L KMnO4 treatment were also beginning to show signs of epithelial lifting from edema and lamellar

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14 fusion. The whole body sections of KMnO4 treated fish appeared to be within normal limits for this species. Discussion The results of this pilo t study suggest that KMnO4 caused more severe gill damage to fish as the salinity increased. A more t horough, replicated experiment will test this hypothesis. The fish in 30 g/L sali nity group treated with 6.0 mg/L KMnO4 died faster than those in the 2 and 15 g/L salinity treatm ents. These results sugge st that fish in the 30 g/L salinity treatment group were mo re severely effected by the KMnO4 treatment. A new experiment will be designe d to test levels of KMnO4 that are not toxic to 100% sailfin mollies (i.e., less than 6 mg/L KMnO4) to determine if KMnO4 causes more severe gill damage and possibly higher mortality at higher salinity levels.

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15 CHAPTER 3 MATERIALS AND METHODS Experimental Procedures Experimental Fish and Quarantine Procedure One thousand five hundred sailfin mollies were obtained from Segrest Farms in Gibsonton, Florida. Upon arri val at the University of Florida Tropical Aquaculture Laboratory (Ruskin, Florida) fish transpor t bags were placed in holding tanks for 30 minutes for temperature acclimation. Fish were exposed to a saltwater bath of 25 g/L for approximately 5 minutes (Stoskopf 1993) to rem ove ectoparasites. Al l salt used in this experiment was made with Instant Ocean sa lt mix (Aquarium Systems, Mentor, Ohio). Fish were weighed in groups of approximate ly 100 (each individual bag with water) on an electronic scale, netted out, and then placed into one of three holding tanks (680 L each, 5 g/L salinity water). The bag and water we re reweighed after fish were released to determine an actual weight of fish in the ba g. Fish were kept in the holding tanks until experimental salinity acclimations (described below) were complete. Any sailfin mollies that died during the salinity acclimation pe riod were necropsied (including microscopic gill, skin, and fin examination) to ensure that the sailfin mollies were remaining as free of parasites as possible and for the early detection of other problems. Fish in the holding tanks were acclimated to water salinities of 2 g/L, 15 g/L, or 30g/L, respectively. A reservoir tank contai ning no fish, but holding approximately 680L of 30 g/L salinity water was used for water ch anges. The water in the reservoir tank was made with Instant Ocean salt mix and wellwater and was aerated for 24 hours prior to

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16 water use (see Appendix B for typical composition). This water was added to the tanks to adjust the salinities upward. Well water was added to decrease salinity. Salinities were verified using a refractometer (Aqua tic Eco-Systems, Apopka, Florida). Fish were fed twice a day, five days a week a generic bulk tricolor flake food (Zeigler Tri-color flake, Gardners, Pennsylvani a). The weight of the total fish population in each holding tank (about 500 fish) was 1247.5 grams (2 g/L salinity water), 1252.5 grams (15 g/L salinity water), and 1249.5 grams (30 g/L salinity water). The approximate average weight per animal was 2.5 grams. Based on those weights, fish were fed 12.5 grams (1% of total body weight in holding tank) of food twice per day, five days a week. Acclimation Procedure Sailfin mollies were divided into one of three different holding tanks (500 fish per tank) that were maintained at 5 g/L salinity. Freshwater was consid ered 2 g/L salinity and the holding tank being adjusted to that sa linity was not adjusted until the seventeenth day of acclimation. The salinity of the othe r two holding tanks was raised twice a week in increments of 5 g/L salinity. During acclimation an active, air-driven, s ponge biofilter was maintained at each of the salinity levels that the fish would be held (10, 15, 20, 25, and 30 g/L) during the acclimation period as a replacement or back-up f ilter. Filters were started 3 weeks before the sailfin mollies arrived; the filters were fed daily with ammonia until they were needed in the fish holding tanks. When the salinity was increased in the holding tanks, the “old” biofilters were replaced with a “new” active biofilter that corresponded with the new salinity of the holding ta nk. Seventeen days after fish a rrival the desired salinities of 2 g/L, 15 g/L, and 30 g/L, respec tively, were reached. Fish were maintained in the holding

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17 tanks for five days more before being distri buted to the treatment tanks. Salinity was monitored daily using a refractometer thr oughout the acclimation pe riod and salinity was adjusted as needed. After acclimation to the appropriate salinity group, the fish were divided into the thirty-six, 75.7-L treatment ta nks filled to 68 liters. Tw elve tanks were randomly assigned to each of the three salinities being tested (2, 15, and 30 g/L). Fish were monitored in the 75.7-L tanks for three days before being exposed to KMnO4. Each tank contained an air-driven sponge filter, whic h also provided aeration. Each tank was stocked with 37 fish, an average biomass of 86.4 grams per tank (fish were reweighed with an electronic scale prior to dispersal). Treatments were assigned to individual tanks using the complete random ized block method (Ott RL and Longnecker M 2001). A reservoir was available for each of the three salinity treatments so that water changes could be done when needed. Response Variables Tanks were checked twice daily for dead fish and results were recorded. Behavior was observed at the following times post KMnO4 treatment: time zero, 0.08, 0.16, 0.33, 0.66, 1.5, 3, 6, 12, 24, 48, 96, and 168 hours. Due to physical constraints, observations for all 36 tanks could not all be made at the same time so a two-minute delay was instated. Spec ifically in applying KMnO4, the first tank was dosed at 9:00 a.m., the second at 9:02 a.m., and so on until a ll tanks had been dosed in succession. Five trained observers were used to score behavior of the experime ntal fish. Behavior scores were categorized as follows: 1. within normal limits – fish sw imming normally in midwater, 2. slight increase in opercular movement,

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18 3. marked increase in opercular movement, fi sh behavior mixed between resting and actively swimming (usually erra tically and repeatedly into the bottom and sides of the tank), 4. beginning to lose the ability to maintain equilibrium or just laying on the bottom of the tank, with obvious, labored respiration, 5. loss of equilibrium, floating throughout the tank with the cu rrent, very slow opercular movement, fish on the verge of death. Tissues for histologic processing were coll ected at three differe nt time points: one hour prior to KMnO4 treatment for controls, twelve hours after initiation of treatment, and seven days post KMnO4 treatment exposure. Six whol e fish per tank were taken for each of the three histological sampling times Prior to tissue collection, fish were euthanized using a 1 g/L dose of buffered (s odium bicarbonate) tricai ne methanesulfonate (Finquel, Argent Laboratories, Redmond, WA) dissolved in water of corresponding salinity, either 2, 15, or 30 g/L. Following euthanasia, a small opening was cut in the fish’s coelom and whole fish were submerge d in 10% neutral buffered formalin for at least 48 hours before processing. Following fixation, all of the gill tissue wa s excised from each fish and placed in individual cassettes for pro cessing. The body of the fixed fi sh was also cut into three transverse sections (or steaks, as in the pilo t study – Chapter 2) and placed in individual cassettes for processing. All cassettes were then placed in decalcif ication solution (CalEX, Fisher Scientific, Pittsburgh, PA) to remo ve any calcified material that could hinder the microtomy of the tissue. The tissue was rinsed in tap water fo r four hours prior to processing. The tissues underwent a fourteen-hour tissue processing including alcohol (7 hours), xylene (3.5 hours), and paraffin wax ( 3.5 hours) – see Appendix A for detailed processing schedule. The tissue was embedded in paraffin. Tissue in the paraffin block

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19 was cut at 5 micrometers and stained using a standard hematoxylin and eosin stain. Slides were ready for viewing by light micros copy. Analysis consisted of scoring each slide by scanning at 100X and examining in de tail at 400X looking at approximately 50 gill filaments total using the following system: 1. Normal, includes background environmenta l damage (inflammation and mucous cell infiltration) at the tip of the gill 2. Secondary lamellar damage including fusion; mucous cell and inflammatory cell infiltration and hyperplasia, damage beginni ng to extend further from the tip of the gill 3. Expansion of lamellar interstitium by edem a and inflammatory cells with lifting off of epithelium as well as fusion of lamellae 4. Necrosis and expansion of la mellar interstitium by edema and inflammatory cells in 50% or more of the lamellae To limit observer bias the labels of each sl ide were covered with a slide label with an arbitrarily assigned number. After all of the slides were scored and recorded, the stickers were removed so that the scores could be matched with the slide iden tification. Experimental Design KMnO4 Treatment The experimental design consisted of expos ing sailfin mollies in each of the three salinities (2, 15, and 30 g/L) to thre e different concentrations of KMnO4, and a control group with no KMnO4 treatment (Table 3-1). The dos ages listed in Table 3-1 are calculated doses. Each treatment was replicated three times, making up 36 treatment tanks. Table 3-1. Experimental treat ment combinations of KMnO4 concentrations and salinity levels. Salinity (g/L) KMnO4 Treatment (mg/L) 2 0, 0.5, 1.0, 3.0 15 0, 0.5, 1.0, 3.0 30 0, 0.5, 1.0, 3.0

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20 Experimental Procedure Salinity was monitored daily using a refract ometer. Fish were not fed during the experiment. During this time 25% water changes were done daily. Potassium permanganate demand was determined for each salinity level using water from randomly selected tanks one and two days prior to the KMnO4 treatment. The resulting six numbers were used to calculate an average KMnO4 demand for all tanks at the corresponding salinity (Table 3-2). Table 3-2. Calculated KMnO4 demand for each salinity level. The mean KMnO4 demand was used to calculate the actual KMnO4 dosage for all treatments. Salinity Level g/L Mean KMnO4 demand mg/L n=6 2 0.34 15 0.36 30 0.41 Three days after the fish had been tr ansferred to the treatment tanks, water chemistry parameters (salinity, pH, TAN, NO2) were tested in each tank. Water chemistry was measured using a HACH Fish Farming kit (model FF-1A, HACH Chemical Company, Loveland, CO) for the 2 g/L salinity tanks and a HACH Marine kit (model FF-3, HACH Chemical Company, Love land, CO) for the 15 and 30 g/L salinity tanks. Water temperature was measured with a mercury thermometer. Water hardness was only tested in make-up water; six 190-L vats of make-up water were made using Instant Ocean saltmix and well water run thr ough a reverse osmosis system (mixing was done with aeration). One hour before KMnO4 treatment, each sponge filter was removed and control histological tissue samples were collecte d. Aeration was continued in each tank throughout the KMnO4 treatment period. Observations were recorded for each replicate

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21 at the following times: time zero, 0.08, 0.16, 0.33, 0.66, 1.5, 3, 6, 12, 24, 48, 96, and 168 hours. Following KMnO4 treatment (12 hours), a 50% water change was done on each tank, followed by the addition of 5 mL of Ko rdon AmQuel (San Francisco, CA) to all tanks (including control ta nks) to deactivate the KMnO4 (as indicated by the AmQuel label). Sponge filters were then repl aced. Daily 25% water changes were done thereafter. Statistics Descriptive statistics of water chemistr y parameters were derived using Minitab version 14.1 (State College, Pennsylvania). A one-way analysis of variance (ANOVA) was performed on the nitrite and unionized ammo nia values by the salinity level followed by Tukey’s HSD multiple comparison procedure. Mortality rate data was analyzed by salinity and the KMnO4 concentration using a two-way ANO VA. The mortality rate data was then analyzed by the salinity, KMnO4 concentration combination using one-way ANOVA followed by Tukey’s HSD multiple comparison procedure to find which treatment concentrations were significant. An arcsin square root transformation of percentage data was performed prior to anal ysis. Behavioral and histological scoring data by the salinity, KMnO4 concentration combination were analyzed using KruskalWallis tests followed by Dunn’s non-parametr ic multiple comparison test. Minitab version 14.1 (State College, Pennsylvania) wa s used for all statistical analysis except Dunn’s multiple comparison test (Hollande r and Wolfe 1973). A Type-I error rate ( ) of 0.05 was used for all analyses except fo r Dunn’s MCP. A Type-I error rate ( ) of 0.15 was used, as recommended by Hollander and Wolfe (1973), for Dunn’s MCP because of the conservative nature of the test.

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22 CHAPTER 4 RESULTS Water Quality Water chemistry parameters varied in each treatment tank. Water quality measurements were taken prior to the KMnO4 application (Table 4-1). The nitrite levels in the 15 g/L salinity treatment group were found to be significantly higher when compared to the 2 and 30 g/L salinity treatment groups (F = 19.45, DF = 2, p < 0.001). The unionized ammonia levels in the 2 g/L salinity treatment group were found to be significantly higher when compared to the 15 and 30 g/L salinity treatment groups (F = 50.35, DF = 2, P < 0.001). Water hardness levels in the reservoir tanks were 256.6 mg/L for 2 g/L salinity, 2,716 mg/L for 15 g/L sali nity, and 5,480 mg/L for 30 g/L salinity (n = 1 for each salinity level). The temperature was 25C in all tanks (n = 36). Table 4-1. Select water chemistry values fo r experimental tanks (n = 12) prior to KMnO4 treatments. Salinity (g/L) pH Unionized Ammonia (mg/L) Nitrite (mg/L) 2 8.33 / 0.0888 0.12 / 0.0435 0.14 / 0.0812 15 8.12 / 0.0389 0.02 / 0.01084 0.89 / 0.363 30 8.15 / 0.0522 0.03 / 0.01311 0.36 / 0.361 Data given in the format of mean / standard deviation. Behavior Fish behavior was affected by exposure to KMnO4. There were significant differences among treatments (H = 30.48, DF = 11, P = 0.001) (Table 4-2). Fish that were significantly impacted showed signs of distress including increased opercular

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23 movement, erratic swimming, beginning loss of equilibrium, and total loss of equilibrium. Table 4-2. Kruskal-Wallis analysis of six-hour behavior scores by treatment combinations (salinity – KMnO4 concentration) (N = 3 tanks per treatment). Treatment Combination: Salinity (g/L) – KmnO4 Concentration (mg/L) Median (see footnote) Average Rank Z 2 – 0.0 0 9.3 -1.57 2 – 0.5 2 13.7 -0.83 2 – 1.0 3 20.0 0.26 2 – 3.0 4 31.5 2.23 15 – 0.0 0 4.0 -2.49 15 – 0.5 3 16.3 -0.37 15 – 1.0 3 23.8 0.92 15 – 3.0 4 31.5 2.23 30 – 0.0 0 5.7 -2.20 30 – 0.5 2 14.7 -0.66 30 – 1.0 3 20.0 0.26 30 – 3.0 4 31.5 2.23 H = 30.48 DF = 11 P = 0.001 (adjusted for ties) Median key: 0 = within normal limits; 2 = marked increase in opercular movement, erratic swimming; 3 = beginning loss of equilib rium, periods of rest on bottom of tank; 4 = total loss of equilibrium. The Dunn’s multiple comparison test demonstr ated that the 2 g/L salinity, 3.0 mg/L KMnO4 treatment (mean rank 27.5), th e 15 g/L salinity, 3.0 mg/L KMnO4 treatment(mean rank = 27.5), and the 30 g/L salinity, 3.0 mg/L KMnO4 treatment (mean rank = 27.5) were significantly different indi cating that the sailfin mollies in those treatment groups lost normal equilibrium and were highly effected by the KMnO4 treatment (Table 4-3). All other treatment groups did not show significance, indicated by a critical value less than 26.3231. Behavior that was affected by exposure to KMnO4 treatment returned to normal within 24 hour s after exposure, including fish exhibiting extreme change.

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24 Table 4-3. Dunn’s multiple comparison test for behavior scores shows significant treatment groups. Treatment Combination: Salinity (g/L) + KMnO4 Concentration (mg/L) Difference in Mean Rank 2 + 3.0 27.5 15 + 3.0 27.5 30 + 3.0 27.5 *A mean rank above the critical value (26.3231) indicates significance when compared to the 15 g/L + 0.0 mg/L KMnO4 concentration (the least e ffected treatment or most normal). The values were found using D unn’s multiple comparison test. All other treatments did not s how significance. Histology The slides of affected gill tissue sh owed secondary lamellar damage including fusion and mucous and inflammatory cell infilt ration and hyperplasia (Figure 4-1). More severely affected gill tissue showed expansion of the lamellar interstitium by edema and inflammatory cell infiltration with lifting off of the epith elium as well as secondary lamellar fusion. The most severe ly affected gill tissue showed severe necrosis as well as expansion of the lamellar interstitium by edema and inflammatory cells with lifting off of the epithelium and secondary lamellar fusion. There were significant differences among treatments (H=51.06, DF=11, P<0.001) (Table 4-4). Significance in th e 15 g/L salinity, 3.0 mg/L KMnO4 indicates expansion of the lamellar interstitium by edema and infl ammatory cells with lifting off of the epithelium as well as secondary lamellar fusi on (Figure 4-2). Signi ficance in the 30 g/L salinity, 3.0 mg/L KMnO4 treatment indicates necrosis of the gill tissue as well as expansion of the lamellar interstitium by edema and inflammatory cells with lifting off of the epithelium and secondary lamellar fusion (F igure 4-3). Slide scores of treated fish returned to normal limits by the 168th hour.

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25 Table 4-4. Kruskal-Wallis analysis of the slide score by the salinity/KMnO4 treatment combinations. Treatment Combination Salinity (g/L) – KmnO4 Concentration (mg/L) N= Median Average Rank Z 2-0 54 0 252.9 -2.66 2-0.5 54 1 268.9 -1.98 2-1.0 54 1 318.7 0.11 2-3.0 54 1 318.7 0.11 15-0 54 1 327.6 0.49 15-0.5 54 1 281.9 -1.44 15-1.0 54 1 348.1 1.37 15-3.0 54 1 391.9 3.20 30-0 54 0 240.5 -3.18 30-0.5 54 1 325.8 0.41 30-1.0 54 1 313.4 -0.11 30-3.0 36 2 446.3 4.41 H = 65.34 DF = 11 P < 0.001 (adjusted for ties) Median key: 0 = within normal limits; 1 = secondary lamellar damage including fusion, mucous and inflammatory cell infiltration and hyperplasia; 2 = secondary lamellar damage including fusion, mucous and inflammato ry cell infiltration and hyperplasia. The 30 g/L salinity, 3.0 mg/L KMnO4 treatment groups shows n=36 due to mortality. This table includes scores from all tissue collection times (zero, 12 hours, and 168 hours). Table 4-5. Dunn’s multiple comparison test fo r histology slide scores shows significantly different treatments. Treatment (g/L salinity – mg/L KMnO4) Significantly Different From: (g/L salinity – mg/L KMnO4) Difference in Mean Rank Critical Value 15 – 3.0 2 – 0.0 139.0 116.535 15 – 3.0 2 – 0.5 123.0 116.535 15 – 3.0 30 – 0.0 151.4 116.535 30 – 3.0 2 – 0.0 193.4 130.290 30 – 3.0 2 – 0.5 177.4 130.290 30 – 3.0 15 – 0.5 164.4 130.290 30 – 3.0 30 – 0.0 205.8 130.290 30 – 3.0 30 – 1.0 132.9 130.290 A mean rank above the critical value (116.535) indicates significance when compared to the treatment indicated. All other treatm ents did not show significance.

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26 A B Figure 4-1. Gills from sailfin mollies in 2 g/L salinity water treated with KMnO4. A(200X, 0.0 mg/L KMnO4). Secondary lamellar structure is well-ma intained (Slide Score = 0). B(200X, 3.0 mg/L KMnO4). Secondary lamellar fusion, as indicated by the arrow, is commonly seen in this treatment group (Slide Score = 1). Stained by H&E. A B C Figure 4-2. Gills from sailfin mollies in 15 g/L salinity water treated with KMnO4. A (200X, 0.0 mg/L KMnO4). The secondary lamellar structure is well-maintained (Slide score = 0). B(200X) and C(400X) (3.0 mg/L KMnO4). There is expansion of the lamellar interstitium by edema and inflammatory cells with lifting off of the epithelium as well as fusion of the lamellae (Slide score = 2). Stained by H&E.

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27 A B C Figure 4-3. Gills from sailfin mollies in 30 g/L salinity water treated with KMnO4. A. (200X, 0.0 mg/L KMnO4). Secondary lamellar structure is well-maintained (Slide Score = 0). B(200X) and C(400X) (3.0 mg/L KMnO4). Necrosis of the lamellae, fusion of the secondary lamellae, as well as lifting of the epithelial layer of cells by expansion of the la mellar interstitium by inflammation and edema was common in this treatment group (Slide score = 3). Stained by H&E. Mortality Salinity and KMnO4 concentration were found to have some significantly different treatment groups (Table 4-6). Cert ain treatment combina tions were also found to be significantly different (Table 4-7). The 30 g/L salinity, 3.0 mg/L KMnO4 treatment had significantly higher mortality than all other treatments, with 100-percent mortality (Figure 4-7). The 15 g/L salinity, 3.0 mg/L KMnO4 treatment had significantly higher

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28 mortality than the 15 g/L salinity, 0.0 and 0.5 mg/L KMnO4 treatments, with 36.33percent mortality (Figure 4-6). The 2 g/L salinity, 3.0 mg/L KMnO4 treatment was not found to be significantly different from ot her treatments, but that treatment had 11.33percent mortality (Figure 4-5). Other tr eatment groups demonstrated very low, nonsignificant mortality over the observation period (168 hours) (see Figures 4.4, 4.5, 4.6, and 4.7). Table 4-6. Two-way ANOVA of the total percentage mortality by the salinity and the KMnO4 concentration. Source Degrees of Freedom Sum of Squares Mean Square F-test P-value Salinity 2 3566.2 1783.08 12.57 < 0.001 KMnO4 Concentration 3 15327.2 5109.06 36.02 < 0.001 Interaction 6 9085.4 1514.23 10.68 < 0.001 Error 24 3404.0 141.83 Total 35 31382.8 Table 4-7. One-way ANOVA analysis of the to tal mortality percentage by the treatment combinations. Source Degrees of Freedom Sum of Squares Mean Square F-test P-value Treatment Combinations 11 6.6463 0.6042 18.62 < 0.001 Error 24 0.7790 0.0325 Total 35 7.4253

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29 Total Percentage Mortality Salinity (g/L) KMnO4 (mg/L) 30 15 2 3.0 1.0 0.5 0.0 3.0 1.0 0.5 0.0 3.0 1.0 0.5 0.0 100 80 60 40 20 0 Figure 4-4. Cumulative Total Percentage Mortal ity Over 7 days by Salinity (fresh 2 g/L, brackish 15 g/L, and salt 30 g/L) and KMnO4 Concentration (0.0, 0.5, 1.0, and 3.0 mg/L). Connected data points = mean. 0 10 20 30 40 50 60 70 80 90 1000.00 0.08 0 .16 0 .33 0 .66 1 .50 3.00 6 .0 0 0 .50 24.0 0 48.0 0 96.00 1 6 8.00Time (hours)Percentage Mortalit y 0.0 mg/L KMnO(sub)4 0.5 mg/L KMnO(sub)4 1.0 mg/L KMnO(sub)4 3.0 mg/L KMnO(sub)4 Figure 4-5. The cumulative total percentage mortality shown over time for the 2 g/L treatments.

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30 0 10 20 30 40 50 60 70 80 90 1000 .00 0 .0 8 0.16 0 .33 0 .66 1 .5 0 3.00 6.00 0 .50 24 .00 4 8.00 96.0 0 1 6 8.00Time (hours)Percentage Mortalit y 0.0 mg/L KMnO(sub)4 0.5 mg/L KMnO(sub)4 1.0 mg/L KMnO(sub)4 3.0 mg/L KMnO(sub)4 Figure 4-6. Cumulative total percentage mortality shown over time for the 15 g/L treatments. 0 10 20 30 40 50 60 70 80 90 1000 0 0 0.08 0. 1 6 0.33 0. 6 6 1 50 3. 0 0 6 0 0 0.50 24 0 0 48.00 96.0 0 168. 0 0Time (hours)Percentage Mortalit y 0.0 mg/L KMnO(sub)4 0.5 mg/L KMnO(sub)4 1.0 mg/L KMnO(sub)4 3.0 mg/L KMnO(sub)4 Figure 4-7. Cumulative total percentage mortality shown over time for the 30 g/L treatments.

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31 CHAPTER 5 DISCUSSION AND CONCLUSIONS Salinity/KMnO4 Treatments Potassium permanganate use in freshwater aquaculture is common. The concentration at which toxic effects are seen is affected by a variet y of factors such as water quality and species specific sensitivity. The effectiveness of KMnO4 is related to the amount of oxidizable material in the water, i.e. organic and inor ganic material, which comprises the KMnO4 demand of the water (Tucker 1984). It has also been suggested that water of high pH can cause manganese dioxide to precipitate (Stuart 1983 and Noga 1996). The purpose of this study was to determine if KMnO4 creates toxicity problems for sailfin mollies held in saltwater compared to cohorts maintained in freshwater. This study examined the effect of KMnO4 on the sailfin molly maintained at different salinity levels and at different KMnO4 concentrations. Tanks treated with 2 g/ L salinity, 3.0 mg/L KMnO4 demonstrated 11.33% cumulative mortality (Figure 4-4) thr oughout the entire 168-hour experiment. Histological results in this experimental group showed only minimal secondary lamellar fusion and mucous and inflammatory cell in filtration and hyperplasia in this same treatment group (Figure 4-1). Sailfin mollies in the 2 g/L salinity water treated with 3.0 mg/L KMnO4 lost equilibrium in the water, dem onstrating that they were negatively affected by the KMnO4 treatment. The 2 g/L treatments also had a high mean ammonia level. Mortality rate in this group may have been affected by the added stress from the high ammonia levels in the water. The 3.0 mg/L KMnO4 concentration is higher than

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32 that of the usual recommended dose of 2.0 mg/L KMnO4. It is possible that the 3.0 mg/L KMnO4 dose is above the safety range of KMnO4 treatment for sailfin mollies kept in 2 g/L salinity water causing minimal mortality. Tanks treated with 3 mg/L KMnO4 compared to control tanks treated with 0.0 mg/L KMnO4, were visually different due to KMnO4 concentration (the higher the KMnO4 concentration the more purple the color of the water). This may cause observers to approximate the KMnO4 concentration level, potential ly creating bias. The behavior scoring system was made in an attempt to a llow the observer to judge the fish solely on their behavior without factoring in the colo r of the water. The combined results of behavior, mortality rate, and histol ogical analysis suggests that KMnO4 treatment may not be a safe application for the 2 g/L salinity, 3.0 mg/L KMnO4 even though it is not significantly different from other treatments. Use of KMnO4 in 15 g/L salinity water is a higher risk treatment than it would be in 2 g/L salinity water. The histology scoring system revealed expansion of the lamellar interstitium by edema and inflammatory cells wi th lifting off of the epithelium as well as fusion of the lamellae. This damage was c onsistently observed in this treatment group (Figure 4-2). The lifting of the epithelium away from the blood vessels presumably results in decreased efficiency of gas ex change and ammonia excretion, and therefore increases the risk of mortality. As the epith elial layer of the lamellae is moved further from the blood vessels more space is create d and it is more difficult for gas (oxygen, CO2) and other metabolites to move through the epithelium into the blood vessel and vice versa. This effect will at least slow gas exchange and at severe levels may stop gas exchange thus killing the fish.

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33 Sailfin mollies in the 15 g/L salinity, 3.0 mg/L KMnO4 demonstrated 36.33% mortality. Mortality vari ability was high (minimum = 0 %, maximum = 79 %), indicating some inconsistency between populati ons housed in different tanks within the same treatment group. This may indicate water chemistry problems (high nitrite levels in the 15 g/L salinity treatments) or that there is variability in how indi vidual fish react to the chemical. A 3 mg/L KMnO4 dose is higher than the recommended dose possibly contributing to toxicity. Further testing with increased replication would be necessary to clarify why there was so much variation in mortality among fish populations in the 15 g/L salinity water, 3.0 mg/L KMnO4 treatment group. As in the 2 g/L salinity, 3.0 mg/L KMnO4, the 15 g/L salinity, 3.0 mg/L KMnO4 treatment groups demonstrated loss of equ ilibrium during the treatment period. In conjunction with high mortality and the gill trauma observe d, this suggests that the 15 g/L salinity water, 3.0 mg/L KMnO4 treatment holds a higher risk for sailfin mollies. KMnO4 should probably not be cons idered as a treatment choice in 15 g/L salinity water without conducting a small-scal e animal safety test on the species being considered. The 30 g/L salinity, 3.0 mg/L KMnO4 treatment was not safe for sailfin mollies in this experiment. As in the 2 and 15 g/L salinity water, 3.0 mg/L KMnO4 treatment groups, the 30 g/L salinity water, 3.0 mg/L KMnO4 treatment group fish lost equilibrium during the treatment. The maximum behavior score of 4 indicated a loss of equilibrium, floating throughout the tank with the current w ith very slow opercular movement. Loss of equilibrium at this point may indicate that the fish is directing all of its energy towards respiration, letting equilibrium take second prio rity, or is so severely debilitated and has lost the ability to maintain its equilibrium.

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34 Histological changes to the gill in th e 30 g/L salinity water, 3.0 mg/L KMnO4 treatment were severe and given the maximum histological score of 3. Lesions included necrosis and expansion of lamellar interstitiu m by edema and inflammatory cells in 50% or more of the lamellae. Gill damage observe d in this group was much more severe than that observed in other treatment groups The response of the gill to KMnO4 treatment, as described above, may have reduced gas excha nge to the point of death by suffocation in fish from this treatment group. Mortality of sailfin mollies in the 30 g/L salinity water, 3.0 mg/L KMnO4 treatment group was significantly different from all ot her treatment groups tested in this experiment. One-hundred percent mortality occurred within 6 hours of exposure to 3.0 mg/L KMnO4 in all replicate groups. The behavior histology, and mortality results from this experiment indicate that KMnO4 is toxic to sailfin mollies from the 30 g/L salinity, 3.0 mg/L KMnO4 treatment. Water Chemistry Parameters Water chemistry analyses among treatment groups were highly variable. Salinity change can cause biological filtration activity to decrease or even discontinue (Hovanec and DeLong 1996). Although an attempt was made to maintain consistent water quality by adding a conditioned biofilter to each of the different salinities encountered by the fish, water quality was variable, especially at the lower salinities. The differences in the nitrification process may have been due to the differences in salinity affecting the rates of bacteria infiltration of the filter. However, the highest mean UIA levels were observed in the 2 g/L salinity and the highest NO2 levels were observed in the 15 g/L salinity. This may have contributed to mortal ity data collected. The stre ss of coping with the water

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35 chemistry problems added to the caustic KMnO4 treatment may have caused mortality in some fish creating variabili ty in the results. Although the 15 g/L salinity, 3.0 mg/L KMnO4 treatment group had 36.33% mortality, the highest mortality was seen in the 30 g/L salinity, 3.0 mg/L KMnO4 treatment. The 30 g/L salinity treatment group exhibited the most normal water quality (in reference to the UIA and NO2 levels), showing that mort ality in this treatment group was probably not related to UIA or NO2 levels. In this experiment the mean pH was 8.33 for 2 g/L salinity, 8.12 for 15 g/L salinity, and 8.15 for 30 g/L salinity, demonstrating low variability. Previous studies (Noga 1996, Stuart 1983) suggested that the high pH of saltwater was respons ible for mortality of fish because manganese dioxide may precipitate ont o the gills. In this study the lack of variation in pH between treatment tanks does not s upport this hypothesis. Ideally pH should have been measured elec tronically instead of colorimetrically. The colorimetric tests lack precision, leading to increased variability. In the future more comprehensive water quality analyses and other methods for parameter detection should be used that do not invol ve colorimetric tests. In this experiment only a calculated dose of KMnO4 was reported. The actual dose of KMnO4 was not tested because the spectroph otometric methods available are not reliable when testing water containing chlo ride or saltwater (D elfino, University of Florida, personal communication). There was no feasible option to determine the actual KMnO4 concentration in all three salinities tested (2, 15, and 30 g/ L, respectively). Technology or methodology should be developed to determine the actual dose of KMnO4 in water regardless of salinity.

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36 Future Research The objective of this experiment wa s to determine toxicity of KMnO4 (0.0, 0.5, 1.0, and 3.0 mg/L) at varying salinity levels (2, 15, and 30 g/L resp ectively) using the sailfin molly as a model. The study demonstrated that a concentration of 3.0 mg/L KMnO4 in water of low organic content was not safe for the sailfin molly in 2, 15 or 30 g/L salinity water as indicated by mean mortality rate s of 11.33, 36.33 and 100 percent, respectively. This data suggests that KMnO4 is not safe for sailfin mollies at concentrations of 3.0 mg/L. The data in this expe riment also suggests that KMnO4 is a higher risk treatment in water with salinity of 15 g/L. The cause of observed KMnO4 related mortality can be speculated to include an unknown reaction between KMnO4 and the salinity, oxidation-reduction by-products that are toxic, or attributes of the sailfin molly that may not be present in other fish. Many elements other than sodium and chloride are present in saltwater. Potassium permanganate may be interacting with one of these elements present in Instant Ocean salt mix (Aquarium Systems, Mentor, Ohio) pr oducing a new compound that causes a toxic reaction in the fish. As indicated by Mallinckrodt Baker (2001), KMnO4 is incompatible with bromides, iodides, ferrous salts, and arse nites, all of which are contained in Instant Ocean salt mix (Aquarium Systems, Mentor, Ohio) in various forms (see Appendix B). The consequences of these interactions ar e unknown in fish, but could be potential hazards. This should be looked into further to rule out other chemical reactions that would result in the fish being exposed to another compound other than KMnO4. One example of a chemical interaction th at could be taking place in saltwater involves KMnO4 and bromide. KMnO4 causes an oxidation-reduc tion reaction, similar to that of ozone (Camel and Bermond 1998). When ozone is used in saltwater systems it

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37 can produce hypobromous acid, hypobromite ion, a nd then bromate through the oxidation of bromide (Tango and Gagnon 2003). These co mpounds may be toxic to fish in the water by way of a pH change. It is po ssible that the oxidation-reduction reaction produced by KMnO4 could have similar byproducts that cause toxicity. In future experiments, pH and oxidation-reduction pot ential should be measured throughout to monitor change. The sailfin molly used in this experiment is a euryhaline species that is able to adapt to differing salinities well. The sailfin mollies’ ability to adapt to fluctuating salinities and ion concentrations may have affected the results of this experiment. The addition of KMnO4 may change the ionic balance of th e water compared to the blood of the fish, impacting osmoregulation. Osmotic hom eostasis must be maintained in the fish to maintain good health. Sailfin mollies may be better suited to living in fresh to brackish water environments as that is more of their natural distribution. It is possible that at higher salinities even tho ugh the sailfin molly is able to surv ive, it is not able to flourish. A sailfin molly in saltwater may be under more environmental stress. Therefore, when challenged with a treatment such as KMnO4, the fish is less able to cope with the added stress. Experiments should be completed to s ee if this possible cause of mortality and gill damage is unique to KMnO4 treatment or if it encompa sses other types of chemical treatment or stressors as well. Conclusions Gill damage such as expansion of lamellar interstitium by edema and inflammatory cells with lifting off of epithelium as well as fusion of lamellae and in severe cases necrosis was seen in the 15 a nd 30 g/L salinity, 3.0 mg/L KMnO4 treatment groups. Mortality was 11.33% in the 2 g/L salinity, 3.0 mg/L KMnO4 treatment group, 36.33%

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38 in the 15 g/L salinity treatment group, 3.0 mg/L KMnO4 treatment group, and 100% in the 30 g/L salinity, 3.0 mg/L KMnO4 treatment suggesting that KMnO4 at the highest concentration tested was not safe for sailfin mollies in this experiment. Results also suggest that as the salin ity of the water increases, the toxicity of KMnO4 to sailfin mollies also increases.

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39 APPENDIX A PROCESSING SCHEDULE FOR THE SH ANDON EXCELSIOR AUTOMATIC TISSUE PROCESSOR Table A-1. Processing schedule for the sh andon excelsior automa tic tissue processor Reagent Used: Time in hours: Alcohol – Ethyl 50-75% 1:00 Alcohol – Ethyl 1:00 Alcohol – Ethyl 1:00 Alcohol – Ethyl 1:10 Alcohol – Ethyl 1:10 Alcohol – Ethyl 75-100% 1:30 Xylene 1:00 Xylene 1:15 Xylene 1:15 Wax – Paraffin 1:00 Wax – Paraffin 1:15 Wax – Paraffin 1:15

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40 APPENDIX B TYPICAL COMPOSITION OF INSTANT OCEAN SALT Table B-1. Typical composition of instant ocean salt Solution at Approximate Salinity of 35ppt Ion Instant Ocean Seawater* (ppm) (ppm) Chloride 19,290 19,353 Sodium 10,780 10,781 Sulfate 2,660 2,712 Magnesium 1,320 1,284 Potassium 420 399 Calcium 400 412 Carbonate/bicarbonate 200 126 Bromide 56 67 Strontium 8.8 7.9 Boron 5.6 4.5 Fluoride 1.0 1.28 Lithium 0.3 0.173 Iodide 0.24 0.06 Barium less than 0.04 0.014 Iron less than 0.04 less than 0.001 Manganese less than 0.025 less than 0.001 Chromium less than 0.015 less than 0.001 Cobalt less than 0.015 less than 0.001 Copper less than 0.0 15 less than 0.001 Nickel less than 0.015 less than 0.001 Selenium less than 0.015 less than 0.001 Vanadium less than 0.015 less than 0.002 Zinc less than 0.0 15 less than 0.001 Molybdenum less than 0.01 0.01 Aluminum less than 0.006 less than 0.001 Lead less than 0.0 05 less than 0.001 Arsenic less than 0.004 0.002 Cadmium less than 0.002 less than 0.001 Nitrate None 1.8 Phosphate None 0.2 Data for seawater values taken from An Introduction to the Chemistry of the Sea 1998. M.E.Q. Pilson

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41 LIST OF REFERENCES American Public Health Association, American Water Works Association, Water Environment Federation: Standard Methods for the Ex amination of Water and Wastewater ed 20, Washington, D.C., 1998. Balsano JS, Rasch EM, and Monaco PJ. 1989. The evolutionary ecology of Poecilia Formosa and its triploid associate. In: Me ffe GK and Snelson FF, editors. Ecology and evolution of livebearing fishes (Poecil iidae). Englewood Cliffs : Prentice Hall. p 277-297. Bishop Y, ed. 2001. The Veterinary Formulary. 5th ed. London : Pharmaceutical Press. 692p. Boyd CE. 1979. Water Quality in Warmwater Fish Ponds. Auburn: Auburn University. 359p. Camel V and Bermond, A. (1998). The use of ozone and associated oxidation processes in drinking water treatment. Water Research 32 : 3208-3222. Carpenter JW, Mashima TY, and Rupiper DJ 2001. Exotic Animal Formulary. 2nd ed. Philadelphia : W.B. Saunders Company. 423p. Carus Chemical Company. 2004. Carus – The World Leader in Potassium Permanganate Technology. http://www.caruschem.com Accessed 10 May 2004. Collins MT, Gratzek JB, Dawe DL, Nemetz TG. 1975. Effects of Parasiticides on Nitrification. Canadian Journal of Fisheries and Aquatic Sciences 32 : 2033-2037. Courtenay WR and Meffe GK. 1989. Small fishes in strange pl aces: a review of introduced poeciliids. In: Meffe GK and Snelson FF, editors. Ecology and evolution of livebearing fishes (Poeciliidae). Englewood Cliffs : Prentice Hall. p 319-332. Darwish AM, Griffin BR, Straus DL, and Mitchell AJ. 2002. Histological and hematological evaluation of potassium pe rmanganate exposure in channel catfish. Journal of Aquatic Animal Health 14 : 134-144.

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42 Engstrom-Heg R. 1971. Direct Measuremen t of potassium permanganate demand and residual potassium permanganate. New York Fish and Game Journal 18 : 117-122. Environmental Protection Agency. 1985. Pes ticide Fact Sheet Number 80. Washington D.C. : Office of Pesticides and Toxic Substances. 3p. Francis-Floyd R and Klinger R. 1997. Use of potassium permanganate to control external infections of ornamental fish. University of Florida Institute of Food and Agricultural Sciences Ex tension Fact Sheet FA-37. Ghosh AK and Pal RN. 1969. Toxicity of four therapeutic compounds to fry of Indian major carps. Fishery Technology VI : 120-123. Gustafson DL. 1981. The influence of salinity on plasma osmolality and routine oxygen consumption in the sailfin molly, Poecilia latipinna (Lesueur), from a freshwater and an estuarine population. Gainesville (FL) : The University of Florida. 24p. Hollander M and Wolfe DA. 1973. Nonparametr ic statistical methods. John Wiley and Sons, New York. Hovanec TA and DeLong EF. 1996. Compara tive analysis of n itrifying bacteria associated with freshwater and marine aquaria. Applied and Environmental Microbiology 62 : 2888-2896. Lawrence JM. 1956. Preliminary results on the use of potassium permanganate to counteract the effects of rotenone on fish. The Progressive Fish-Culturist 18 : 1521. Lay BA. 1971. Applications for potassi um permanganate in fish culture. Transactions of the American Fisheries Society 100 : 813-816. Levine G and Meade TL. 1976. The Effects of Disease Treatment on Nitrification in Closed System Aquaculture. In: World Ma riculture Society, editor. Proceedings of the seventh annual meeting, World Mari culture Society. San Diego, California, January 25-29, p. 483-491. Mallinckrodt Baker, Inc. 2001. Material Sa fety Data Sheet – Potassium Permanganate. Mallinckrodt Chemicals / J.T. Baker website. http://www.jtbaker.com/msds/englishhtml/p6005.htm Accessed 2004 May 10. Marking L and Bills T. 1975. Toxicity of potassium permanganate to fish and its effectiveness for detoxifying antimycin. Transactions of the American Fisheries Society 104 : 579-583. Masser MP and Jensen JW. 1991. Calculati ng treatments for ponds and tanks. Southern Regional Aquaculture Cent er Publication No. 410.

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43 Meffe GK and Snelson FF. 1989. An ecological overview of poecilid fishes. In: Meffe GK and Snelson FF, editors. Ecology and evolution of livebearing fishes (Poeciliidae). Englewood Cliffs : Prentice Hall. p 13-32. Noga EJ. 1996. Fish Disease: diagnosis a nd treatment. St. Louis : Mosby-Year Book, Inc. 367p. Nordlie FG, Haney DC, and Walsh SJ. 1992. Comparisons of salinity tolerance and osmotic regulatory capabilities in populations of sailfin molly ( Poecilia latipinna ) from brackish and fresh waters. Copeia 1992 : 741-746. Ott RL and Longnecker M. 2001. An introdu ction to statistical methods and data analysis. Pacific Grove: Duxbury. 1152p. Parenti LR and Rauchenberger M. 1989. Sy stematic overview of the poeciliines. In: Meffe GK and Snelson FF, editors. Ecol ogy and evolution of livebearing fishes (Poeciliidae). Englewood Cliffs : Prentice Hall. p 3-12. Petrovicky I. 1988. Aquarium fish of the world. London : The Hamlyn Publishing Group Limited. 499p. Plumb JA. 1999. Health maintenance and princi pal microbial diseases of cultured fishes. Ames : Iowa State University Press. 328p. Reardon IS and Harrell RM. 1994. Effects of varying salinities on the toxicity of potassium permanganate to larval and juvenile striped bass, Morone saxatilis (Walbaum). Aquaculture and Fisheries Management 25 : 571-578. Robins R. 2003 Oct 20. Biological profile of the sailfin molly. Florida Museum of Natural History website. http://www.flmnh.ufl.edu/fish/Gallery/D escript/SailfinMolly /SailfinMolly.html Accessed 2004 May 6. Spotte S. 1992. Captive seawater fishes : science and technology. New York : John Wiley & Sons, Inc. 942p. Stoskopf MK. 1993. Fish Medicine. Philadelphia : W.B. Saunders Company. 882p. Straus DL and Griffin BR. 2002. Efficacy of potassium permanganate in treating ichthyophthiriasis in channel catfish. Journal of Aquatic Animal Health 14 : 145148. Stuart N. 1983. Treatment of Fish Diseases. The Veterinary Record 112 : 173-177. Tango MS and Gagnon GA. 2003. Impact of ozonation on water quality in marine recirculating systems. Aquaculture Engineering 29 : 125-137.

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44 Thomas-Jinu S and Goodwin AE. 2004. Acute columnaris infection in channel catfish, Ictalurus punctatus (Rafinesque): efficacy of prac tical treatments for warmwater aquaculture ponds. Journal of Fish Diseases 27 : 23-28. Tucker CS. 1984. Potassium permanganate demand of pond waters. Progressive FishCulturist 46 : 24-28 Tucker CS. 1989. Method for estimating po tassium permanganate disease treatment rates for channel catfish. The Progressive Fish-Culturist 51 : 24-26. Tucker CS and Boyd CE. 1977. Relations hip between potassium permanganate treatment and water quality. Transactions of the Amer ican Fisheries Society 106 : 481-488.

PAGE 55

45 BIOGRAPHICAL SKETCH Emily N. Marecaux grew up in Ashland, Ma ine. After graduating from high school she attended college at The University of Findlay in Findlay, Ohio. While at The University of Findlay she pursued a Bachelor of Science degree majoring in biology, preveterinary science, with a minor in chem istry. After graduating in May of 2002, Emily came to The University of Florida to work on a Master of Science degree from the Department of Fisheries and Aquatic Sciences with a focus on fish health. Emily has accepted a position at the University of Arkansas at Pine Bluff as a fish health extension associate.


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Copyright Date: 2008

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EFFECTS OF POTASSIUM PERMANGANATE ON THE SAILFIN MOLLY, Poecilia
latippinna, AT VARYING SALINITY LEVELS















By

EMILY N. MARECAUX


A THESIS PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
MASTER OF SCIENCE

UNIVERSITY OF FLORIDA


2006

































Copyright 2006

by

Emily N. Marecaux


























To my family and friends who have supported me throughout my college career.














ACKNOWLEDGMENTS

My committee members, Ruth Francis-Floyd, B. Denise Petty, Scott P. Terrell,

Kathleen H. Hartman, and Roy P. E. Yanong, have provided significant contributions to

this project. Jeff Hill provided extremely important help with the statistical analysis of

this project. Sherry Giardina provided helpful guidance through the graduate student

process. Scott Graves of the University of Florida Tropical Aquaculture Laboratory

helped me set up the aquaculture tank system used for this project. Jamie Holloway from

the Department of Fisheries and Aquatic Sciences helped with the pilot study and

gathering of supplies for this project. Patricia Lewis and Don Samuelson from the

University of Florida College of Veterinary Medicine allowed for the use of the histology

laboratory which was an integral part of my project. Robert Leonard, Tina Crosby, Jen

Matysczak helped gather behavioral observation data. Chris Langeneck took the pictures

of the sailfin mollies. Finally, the generous support from Segrest Farms was very much

appreciated and the project could not have been completed without their donation. Thank

you to everyone that helped this project come to completion.
















TABLE OF CONTENTS



A C K N O W L E D G M E N T S ................................................................................................. iv

LIST OF TABLES .......... .......................................... ................ ........ vii

LIST OF FIGURE S ........ ........ .......................................... .............. viii

ABSTRACT .............. .................. .......... .............. ix

CHAPTER

1 L ITER A TU R E R E V IEW ......... ................... ......... ........................................1

Introduction ................. .................. ...........1....
The Sailfin Molly Poecilia latipinna........................................... .................. 3
Potassium Permanganate KMnO4...................................... .. ............... 5
T ables and F figures ................................................................... .......................... . 9

2 PILO T STU D Y ........... ............................... ...... ...... .............. .. 11

E xperim mental D design ........................................................ ........ .. .... ........ 11
R e su lts .................................................................................................................... 1 3
D isc u ssio n .............................................................................................................. 1 4

3 M ATERIALS AND M ETHOD S ........................................... .......................... 15

Experim ental Procedures ......... .......................................... ....... ...............15
Experimental Fish and Quarantine Procedure.................................. ...............15
Acclimation Procedure ............................ ......... .. ..... ............... 16
R espon se V ariables ............................ ........................ .. ....... .... ............17
Experim mental D design ................................. ......... .......... ....... 19
KM nO 4 Treatm ent ........... .. ................................. .... .......... ...... .... 19
E xperim ental P procedure ........................................ .........................................20
Statistics ..................... ............ .............................. 21

4 R E S U L T S .............................................................................2 2

W after Q uality................................................. 22
Behavior ......................... ..............................22



v









H isto lo g y ........................................................................................2 4
M o rta lity ..............................................................................2 7

5 DISCUSSION AND CONCLUSIONS ............... ......... ................................31

APPENDIX

A PROCESSING SCHEDULE FOR THE SHANDON EXCELSIOR
AUTOMATIC TISSUE PROCESSOR.................................. ........................ 39

B TYPICAL COMPOSITION OF INSTANT OCEAN SALT....................................40

L IST O F R E F E R E N C E S ........................................................................ .....................4 1

B IO G R A PH IC A L SK E TCH ..................................................................... ..................45
















LIST OF TABLES


Table page

1-1 The 96h LC5o levels for potassium permanganate (KMnO4) determined for
juvenile and larval striped bass, Morone saxatilis. ...............................................10

1-2 Dosages and target pathogens of KMnO4 based on literature review........ ........ 10

3-1 Experimental treatment combinations of KMnO4 concentrations and salinity
le v e ls ............................................................................ ................ 1 9

3-2 Calculated KMnO4 demand for each salinity level ...........................................20

4-1 Select water chemistry values for experimental tanks (n = 12) prior to KMnO4
treatm ents. ...........................................................................22

4-2 Kruskal-Wallis analysis of six-hour behavior scores by treatment combinations
(salinity KMnO4 concentration) (N = 3 tanks per treatment).............................23

4-3 Dunn's multiple comparison test for behavior scores shows significant treatment
group p s. .............................................................................. 24

4-4 Kruskal-Wallis analysis of the slide score by the salinity/KMnO4 treatment
com bination s. ...................................................... ................. 2 5

4-5 Dunn's multiple comparison test for histology slide scores shows significantly
different treatm ents. ...................... .................. ... .... ................. 25

4-6 Two-way ANOVA of the total percentage mortality by the salinity and the
K M nO 4 concentration. ..................................................................... .................. 28

4-7 One-way ANOVA analysis of the total mortality percentage by the treatment
com bination s. ...................................................... ................. 2 8

A-1 Processing schedule for the shandon excelsior automatic tissue processor ............39

B-l Typical com position of instant ocean salt ............................................................. 40
















LIST OF FIGURES


Figure p

1-1 The sailfin m olly .................. .. .. ............................................ .... .9

4-1 Gills from sailfin mollies in 2 g/L salinity water treated with KMnO4 ................26

4-2 Gills from sailfin mollies in 15 g/L salinity water treated with KMnO4................26

4-3 Gills from sailfin mollies in 30 g/L salinity water treated with KMnO4................27

4-4 Cumulative Total Percentage Mortality Over 7 days by Salinity (fresh 2 g/L,
brackish 15 g/L, and salt 30 g/L) and KMnO4 Concentration (0.0, 0.5, 1.0, and
3.0 m g/L) ................................ ................................. ........... 29

4-5 The cumulative total percentage mortality shown over time for the 2 g/L
treatm ents. ...........................................................................29

4-6 Cumulative total percentage mortality shown over time for the 15 g/L
treatm ents. .......................................................................... 30

4-7 Cumulative total percentage mortality shown over time for the 30 g/L
treatm ents. .......................................................................... 30















Abstract of Thesis Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Master of Science

EFFECTS OF POTASSIUM PERMANGANATE ON THE SAILFIN MOLLY, Poecilia
latipinna, AT VARYING SALINITY LEVELS

By

Emily N. Marecaux

May 2006

Chair: Ruth Francis-Floyd
Major Department: Fisheries and Aquatic Sciences

Potassium permanganate (KMnO4) is used in fish culture for disease treatment,

water clarification, rotenone detoxification, and historically for management of oxygen

depletion. Most commonly, KMnO4 is used in freshwater systems at 2 mg/L to control

ectoparasites, bacteria, and fungi. Effective concentrations are determined by the KMnO4

demand of the water being treated. Although safe use of KMnO4 in freshwater systems is

well documented, its toxicity to fish in saltwater systems is less well known.

The sailfin molly, Poecilia latipinna, a euryhaline species, was used as a model to

test the toxicity of KMnO4 at varying concentrations and at different salinity levels.

Target KMnO4 concentrations of 0.0, 0.5, 1.0, and 3.0 mg/L plus the KMnO4 demand

were tested. Toxicity was tested at salinity levels of 2, 15, and 30 g/L. Mortality rates

and fish behavior were monitored throughout the experiment and tissue samples for

histological analysis were taken at time zero, immediately post-treatment (12 hours), and

at the end of the monitoring period (7 days).









The mortality rate was significantly higher in the 30 g/L salinity, 3.0 mg/L KMnO4

treatment group than in any other treatment group (p < 0.001). The 15 g/L salinity, 3.0

mg/L KMnO4 treatment group was also found to be significantly different from the 15

g/L salinity 0.0 and 0.5 mg/L KMnO4 treatment groups (p < 0.001). The 2 g/L salinity,

3.0 mg/L KMnO4 treatment was not found to be significantly different. Dunn's multiple

comparison test indicated that treatments of 2 g/L, 15 g/L, and 30 g/L salinities treated

with 3.0 mg/L showed significant changes in behavior resulting in the loss of

equilibrium. Dunn's multiple comparison test also indicated that treatments 15 and 30

g/L salinity, 3.0 mg/L KMnO4 concentration showed significantly different gill damage

as indicated by secondary lamellar fusion, lifting of epithelial cell lining by expansion of

the lamellar interstitium by inflammation and edema, and necrosis.

Results from this study suggest that KMnO4 at concentrations of 0.5 and 1.0 mg/L

may be safe for use in water containing sailfin mollies in water of salinities of 2, 15, and

30 g/L. However, KMnO4 should not be used at concentrations of 3.0 mg/L in 2, 15 or

30 g/L salinity water on the sailfin molly until further research is conducted. Toxicity of

potassium permanganate increased in the higher salinity groups (15 and 30 g/L)

compared to the low salinity group (2 g/L).














CHAPTER 1
LITERATURE REVIEW

Introduction

Potassium permanganate, KMnO4, is an oxidizing agent that is used in fish culture

for disease treatment, water clarification, rotenone detoxification, and historically for

management of oxygen depletion in fish ponds. Potassium permanganate has been used

to treat external pathogens including fungus, bacteria, and some parasites (Lay 1971;

Masser and Jenson 1991; Noga 1996; Francis-Floyd and Klinger 1997; Plumb 1999;

Carpenter et al. 2001; Stoskopf 1993; Bishop 2001; Straus and Griffin 2002; Thomas-

Jinu and Goodwin 2004). KMnO4 can also be used for water clarification purposes (Lay

1971) by oxidizing organic material in the water, forming precipitates that can be

removed from the water by a filter. When rotenone is used in water treatments, KMnO4

can be used as a counter agent to detoxify the water (Lawrence 1956). KMnO4 was also

used to add oxygen to aquaculture ponds in situations of low dissolved oxygen levels in

water (Lay 1971). This method is no longer practiced as KMnO4 did not raise oxygen

levels significantly and may cause oxygen levels to decrease by killing the oxygen

producing algae in the pond (Tucker and Boyd 1977).

Many different treatment regimes have been developed over the years for the

different uses of KMnO4 in aquaculture. However, use of KMnO4 in salt water is less

common than in freshwater systems so less is known about what happens when it is used

as a treatment in the marine environment. There is debate over the use of KMnO4 for









treatment of marine fish and concern regarding the toxicity threshold to KMnO4 in

marine species held in saltwater.

Marking and Bills (1975) found that KMnO4 was more toxic to fish in waters with

pH between 8.5 and 9.5 and total hardness of 300 mg/L as CaCO3 (compared to water

with a total hardness of 12 mg/L as CaCO3). Stuart (1983) and Noga (1996) have

suggested that KMnO4 is toxic to fish in saltwater because of the higher pH typically

associated with saltwater, which may cause manganese dioxide to precipitate onto the

gills. Natural seawater typically has a pH between 7.8 and 8.2 (Spotte 1992), which may

be higher than the pH of some freshwater systems, which are commonly between 6.8 and

7.2. In a freshwater efficacy study using KMnO4 at a dosage of up to 1.5 mg/L to treat

ichthyophthiriasis in channel catfish, Ictaluruspunctatus, the pH was maintained at 8.5

+/- 0.1 (Straus and Griffin 2002), and the authors reported that no fish died in the

effective treatment group (1.25 mg/L). This suggests that pH levels above those typically

found in freshwater may not be the sole cause of KMnO4 toxicity to fish.

Reardon and Harrell (1994) determined the KMnO4 concentration that caused

50% mortality of exposed population over 96 hours, 96h LC5o, at different salinity levels

up to 15 g/L for juvenile and larval striped bass, Morone saxatilis. In this study juveniles

were most tolerant of KMnO4 at a salinity level of 5 g/L, while larvae were most tolerant

of KMnO4 at a salinity level of 3 g/L. Both juvenile and larval striped bass were least

tolerant of KMnO4 at salinity levels of zero. The 96h LC5o levels that Reardon and

Harrell (1994) reported are depicted in Table 1-1 (see page 10).

There was a significant decrease in the 96h LC5o level at salinity levels of 0, 10,

and 15 g/L compared to the 5 g/L treatment for juveniles with KMnO4 being most toxic









to juveniles at the 0 g/L salinity followed by the 15 g/L salinity. It was suggested that the

greatest toxicity occurred in 0 g/L water because this was the salinity where the greatest

osmotic imbalance occurred. This experiment showed that margin of animal safety may

narrow when KMnO4 is applied at higher salinities, but it did not demonstrate if the trend

continued as salinity increased above 15 g/L.

The Sailfin Molly Poecilia latipinna

The sailfin molly, Poecilia latipinna, formerly described and named Mollienesia

latipinna by Charles Alexandre Lesueur in 1821 (Robins 2003), is from the family

Poeciliidae, comprising over 190 species (Parenti and Rauchenberger 1989). The natural

distribution of the sailfin molly is fresh, brackish, and salt waters of Florida, Mexico,

Texas, South and North Carolina, and Virginia (Petrovicky 1988; Robins 2003;

Courtenay and Meffe 1989). Non-indigenous populations are established in the western

U.S. (Arizona, California, and Nevada), Hawaii, Canada, Central America, Singapore,

Australia, New Zealand, Guam, and the Philippines (Courtenay and Meffe 1989). The

sailfin molly prefers lowland areas such as marshes, lowland streams, swamps, and

estuaries (Robins 2003).

The sailfin molly is a fusiform shaped small fish (15-53 mm total length) with a

small head and upturned mouth (Robins 2003)(see Figure 1-1, page 9). The dorsal fin is

greatly enlarged in mature males compared to those of mature females. The dorsal fin is

used as a display to attract females for reproduction. Only dominant males display the

dorsal fin. Subordinate males use the "ambush" technique for breeding (Balsano et al.

1989). The "ambush" technique refers to the chance that the dominant male becomes

distracted, allowing the subordinate male to breed with the female before being chased

away. Males have a modified anal fin called the gonopodium, which is used for internal









fertilization. At rest the gonopodium points caudally, but during reproduction, the

gonopodium is pointed forward and is inserted into the female in a quick motion, which

results in sperm being deposited into the female. The female molly can store the sperm

deposited by the male. The gestation period is 3-4 weeks. Females are viviparous and

give birth multiple times during the year (Robins 2003).

The sailfin molly, Poecilia latipinna, is a euryhaline species that can tolerate

salinities as high as 70 to 80 g/L (Nordlie et al. 1992). Gustafson (1981) used short-term

salinity acclimation as a method to evaluate the influence of salinity on plasma

osmoregulation and routine oxygen consumption. Gustafson (1981) altered the salinity at

a rate of 4.14 g/L per day for sailfin mollies of brackish water origin (mostly 10-20 g/L,

but ranged from 4-35 g/L) and for sailfin mollies of freshwater origin (no information on

the salinity level given). Frank Nordlie (The University of Florida, personal

communication) recommended salinity level adjustments at a rate of 5 g/L every five

days for proper osmoregulation balance. Sailfin mollies are highly adaptable to changing

salinity ranges that are found in their natural habitat.

For aquaculture purposes, salinity up to 3 g/L is considered freshwater (Chapman,

The University of Florida, personal communication), while brackish water ranges from

3.0 g/L to 29 g/L and saltwater is 30 g/L and above. Some species are more sensitive to

salinity than others, therefore, it is necessary to know the limits of the species being used

for experimentation.

The sailfin molly is primarily an herbivore, eating plants and algae, but is also

opportunistic and will eat other food items including detritus or insect larvae and

cannibalism has been reported (Meffe and Snelson 1989). The sailfin molly is a prey









item for many predators. Predators that eat this fish include reptiles, birds, other fishes,

amphibians, and insects. The sailfin molly is also popular in the aquarium trade and is

available in a wide variety of colors through domestication. It has also been used for

research and for biological control of mosquitoes (Courtenay and Meffe 1989).

Potassium Permanganate KMnO4

Historically, the Environmental Protection Agency (EPA) (1985), registered

KMnO4 for use in cooling towers, evaporative condensers, air wash systems, ornamental

ponds, cooling fountains, aquaria, human drinking water, poultry drinking water, for

surface disinfection, sanitization, and as a deodorizer. However, KMnO4 is now exempt

from registration by the EPA because it does not pose unreasonable risks to public health

or the environment.

Currently KMnO4 is a U.S. Food and Drug Administration (FDA), investigational

new animal drug (INAD) under investigation by Carus Chemical Company (Peru, IL)

and Stuttgart National Aquaculture Research Center (SNARC) (Stuttgart, AR). FDA

approval will allow for the legal use of KMnO4 in water containing food fish. To gain

approval from the FDA, Carus Chemical Company is responsible for research on product

chemistry and mammalian toxicology, while SNARC is responsible for research on

efficacy, human food safety, target animal safety, and environmental safety (Straus,

SNARC, personal communication). Regulatory action on KMnO4 has been deferred

pending the outcome of current research.

As a therapeutant for fish, KMnO4 has been used as an external bactericidal and

fungicidal agent. Formulations of KMnO4 are available as ready-to-use liquids, pellets or

tablets, powder, or crystals. The active ingredient is the permanganate ion. It functions

as a strong oxidizing agent that is corrosive and burns any organic material it comes into









contact with. For this reason it is effective if used in fish culture to control external

disease-causing agents including bacteria, fungi, and some parasites. The oxidizing

activity is also the primary problem for treated fish.

As an oxidizing agent, KMnO4 is able to add oxygen, remove hydrogen, or

remove electrons from an element or compound (Cams Chemical Company 2004). For

example in drinking water treatments, KMnO4 is able to oxidize soluble manganese and

iron into manganese dioxide and iron oxide, which are insoluble and can be removed by

filtration.

The effectiveness of KMnO4 is related to the amount of oxidizable material in the

water, i.e. organic material and other elements (inorganic) that may be oxidized. This is

referred to as the KMnO4 demand of the water (Tucker 1984). Engstrom-Heg (1971)

developed a test, using a spectrophotometer, to determine the KMnO4 demand of the

water to be treated. Later, Boyd (1979) developed a quick visual test to determine the

KMnO4 demand of the water to be treated. He treated 1,000 mL samples of water with 0,

1, 2, 3, 4, 6, 8, 10, and 12 mg/L of KMnO4 in separate containers; after fifteen minutes,

the container with the lowest concentration that was still pink was considered the KMnO4

demand of the water.

Potassium permanganate is rendered inactive by organic material (Tucker and

Boyd 1977), therefore, in a recirculating system the biological filter may be affected

because organic material, including the nitrifying bacteria, may be oxidized. However, it

is unclear if KMnO4 has a significant effect on the efficiency of the biofilter (Spotte

1992). One study (Levine and Meade 1976) demonstrated that a treatment of KMnO4

inhibited nitrification 86%, while another study (Collins et al. 1975) demonstrated that









KMnO4 had no effect on nitrification. If the filter has a build-up of organic material, it

will increase the KMnO4 demand of the water if the filter is left online during treatment

and the biological portion of the filter may be damaged.

Tucker (1989) developed a method to estimate the required treatment of KMnO4

based on the 15-minute KMnO4 demand of the water. The calculation from his work is

2.5 multiplied by the value obtained in the fifteen-minute test. However, the level of

KMnO4 needed to control an ichthyophthiriasis outbreak in an efficacy study was 1.25

mg/L (Straus and Griffin 2002). In that study it was determined that using Tucker's

method, the treatment rate indicated would be 1.0 mg/L. This implies that Tucker's

method is not a "fail-safe" method for determining treatment dosages. Another

recommendation for compensation of the KMnO4 demand is to add 2 mg/L to the

KMnO4 demand of the water (Plumb 1999). Other treatment recommendations found in

the literature are summarized in Table 1-2 (see page 10).

The toxicity margin of KMnO4 is narrow (1-3 mg/L) (Plumb 1999). Toxicity

levels have been determined for carp fry as an LC5o ranging from 37.5 to 48 mg/L at

26C and 45 to 37.5 mg/L at 320C, at 24 and 48 hours, respectively (Ghosh and Pal

1969). The pH was maintained between 7.8 and 8.2. Studies have shown that 20 mg/L

KMnO4 is toxic to guppies and 3.2 mg/L KMnO4 is toxic to catfish (Scott and Warren as

cited by Lay 1981), however KMnO4 demand or exposure time is not reported.

Lawrence (1956) reported that toxicity levels of KMnO4 were 3 mg/L for bluegills, 4

mg/L for largemouth bass, and 6 mg/L for goldfish. Finally Lawrence (1956) showed a

toxicity level of 5 mg/L KMnO4 for fathead minnows at a temperature of 680F in a

prolonged bath (no method for removing the KMnO4 was employed), but restocking the









aquaria 24 hours after treatment showed that the water was no longer toxic to fish at that

time. Toxicity of KMnO4 decreases with increasing KMnO4 demand (Tucker and Boyd

1977). Potassium permanganate tolerance is dependent on water quality, exposure time,

and the species of fish.

Potassium permanganate efficacy studies have been conducted with columnaris

and ichthyophthiriasis. Straus and Griffin (2002) reported that a treatment of 1.25 mg/L

KMnO4 was effective against ichthyophthiriasis, meaning that no trophonts were found

on treated channel catfish, Ictaluruspunctatus. In that study, treatments were

administered daily after a complete water change. The pH was maintained at 8.5 +/- 0.1

with a temperature of 170C +/- 2.5 and the study was run until the control fish died. In a

challenge against columnaris, KMnO4 applied at a dose of 2 mg/L as an indefinite bath at

a temperature of 220C, reduced mortality to 69% compared to the 100% mortality in

infected control channel catfish, Ictaluruspunctatus (Thomas-Jinu and Goodwin 2004).

A fish that has been treated with KMnO4 may show signs of tissue damage

depending on the dose ofKMnO4. Darwish et al. (2002) found that channel catfish,

Ictaluruspunctatus, treated with KMnO4 (0.438, 1.315, and 2.190 mg/L) at a pH of 7 +/-

0.2, showed microscopic lesions in the gills using a routine hematoxylin and eosin

histology stain, while liver and trunk kidney showed no lesions. The fish treated with

0.438 mg/L KMnO4 showed mild hypertrophy and spongiosis of the epithelium of the gill

filaments and lamellae. The fish treated with 1.315 and 2.190 mg/L KMnO4 showed

extensive gill epithelial hyperplasia, lamellar fusion, and obliteration of the interlamellar

spaces with inflammatory exudates containing necrotic epithelial cells.









Potassium permanganate is a strong oxidizer and contact with other materials may

cause fire; however by itself it is stable and will keep indefinitely if stored correctly.

Potassium permanganate is incompatible with the following substances: formaldehyde,

powdered metals, alcohol, arsenites, bromides, iodides, phosphorous, sulfuric acid,

organic compounds, sulfur, activated carbon, hydrides, strong hydrogen peroxide, ferrous

or mercurous salts, hypophosphites, hyposulfites, sulfites, peroxides, and oxalates. Care

should also be taken when handling potassium permanganate; it is corrosive and may

cause bums to any area of contact and may be harmful if swallowed or inhaled.

(Mallinckrodt Baker, Inc. 2001)

Potassium permanganate is an important chemical in aquaculture because of its

availability and its various uses. The goal of this study is to determine ifKMnO4 creates

toxicity problems for sailfin mollies held in saltwater compared to cohorts maintained in

freshwater. This study will examine the effect of KMnO4 on the sailfin molly maintained

at different salinity levels and at different KMnO4 concentrations.

Tables and Figures


Figure 1-1. The sailfin molly. A. The gonopodium on this male sailfin molly is
indicated by the arrow. B. A group of sailfin mollies. Photo credit: Chris
Langeneck.










Table 1-1. The 96h LC5o levels for potassium permanganate (KMnO4) determined for
juvenile and larval striped bass, Morone saxatilis.
Juveniles (One-month old) Larvae (18-day old)
Salinity Level 96h LC50 Level Salinity Level 96h LC50 Level
g/L mg/L KMnO4 g/L mg/L KMnO4
0 0.96a 0 1.02a
5 3.26b 3 2.11b
10 1.63c 6 1.41ab
15 1.48c 9 1.73ab
*Data taken from Reardon and Harrell (1994). Means with identical superscript are not
significantly different at the 5% level using the SNK test.

Table 1-2. Dosages and target pathogens of KMnO4 based on literature review.
Recommended dose Duration of
of KMnO4 (mg/L) treatment Treatment Use References
1000 10-40 second fungi, protozoa Carpenter et al. 2001;
bath Noga 1996; Lay 1971

100 5-10 minute fish lice Carpenter et al. 2001;
bath Noga 1996

20 1 hour crustaceans, Stoskopf 1993
protozoa
10 30 minutes fungi, protozoa Lay 1971

5 30-60 minute ectoparasites, Carpenter et al. 2001;
bath gill/skin bacterial Noga 1996
infections

2 prolonged ectoparasites, Bishop 2001; Noga
immersion or gill/skin bacterial 1996; Thomas-Jinu and
bath, infections, Goodwin 2004; Francis-
indefinite, at protozoa, Floyd and Klinger
least 4 hours, oxidation/ 1997; Plumb 1999;
or at least 12 detoxification of Masser and Jensen 1991
hours hydrogen sulfide

2-5 indefinite crustaceans, Stoskopf 1993
protozoa
2 prolonged counteract Lawrence 1956
bath rotenone

2.5 flush for 4 bacterial gill Stoskopf 1993
consecutive disease in
days salmonids
2 flush ectoparasites, Noga 1996
gill/skin bacterial
infections














CHAPTER 2
PILOT STUDY

Experimental Design

A pilot study was conducted at the University of Florida Department of Fisheries

and Aquatic Sciences wet lab (Gainesville, Florida) to determine KMnO4 treatment levels

to be tested. Six, 37.85L tanks (filled to 30 L), each with one airstone, were set up at a

salinity level of 5 g/L. Salinity was tested using a refractometer (Aquatic Eco-Systems,

Apopka, Florida). Thirty-six sailfin mollies, Poecilia latipinna, (mean weight = 2.3

grams) were received from Segrest Farms (Gibsonton, Florida) and six fish were

arbitrarily placed into each tank. Fish were fed once daily (1% of total biomass of tank) a

flake fish food (Zeigler Tri-color flake, Gardners, Pennsylvania). Fifty percent water

changes were conducted every other day in conjunction with salinity adjustments.

Saltwater was made with Instant Ocean salt mix (Aquarium Systems, Mentor,

Ohio). Salinity was adjusted so that two tanks each were established at 2 g/L, 15 g/L, and

30 g/L, respectively, over a time period of fourteen days. In an effort to meet a deadline,

the salinity level was altered 5 g/L every other day until the desired salinity was reached,

with the exception of the 2 g/L tanks, which were adjusted by an increment of 3 g/L. The

salinity level of 2 g/L was chosen over 0 g/L because captive sailfin mollies prefer to live

in water that contains some salt (Petty, University of Florida, personal communication).

The treatment level of KMnO4 to be tested was 2 mg/L and 6 mg/L; one tank of each

concentration (2 and 6 mg/L) was represented at each salinity (2, 15, and 30 g/L).









The KMnO4 demand of the water was determined to be 0.20 mg/L KMnO4 for 30

g/L salinity water, 0.15 mg/L KMnO4 for 15 g/L salinity water, and 0.10 mg/L KMnO4

for 2 g/L salinity water. Actual concentrations ofKMnO4 were not determined because

chloride interferes with typical spectrophotometric techniques that might be used

(Delfino, The University of Florida, 2004) because it reacts with KMnO4. Each salinity

would require its own standardization curve and the higher salinity water would produce

a less reliable curve because the active concentration of KMnO4 would be changing with

increased contact time (some of the inorganic compounds, including chloride, of

saltwater reduce KMnO4).

The 16th day after fish arrival a fifty percent water change was done on each tank

and one fish was sampled from each tank for histologic assessment of tissue prior to

KMnO4 exposure. The designated KMnO4 treatment (either 2 or 6 mg/L KMnO4) plus

the KMnO4 demand was added to the tanks and behavior and mortality was monitored for

twelve hours. Behavioral observations were recorded to aid in the design of the scoring

system for the larger experiment at time zero (0), 0.08, 0.16, 0.33, 0.66, 1.5, 3, 6, and 12

hours. Each tank was dosed with KMnO4 and then fish were observed for behavioral

alterations for 2 minutes until all tanks had been observed at this observation time. This

required two people to physically monitor the tanks for the first four time periods (zero,

0.08, 0.16 and 0.33 hours). All fish were sampled for histological analysis either at the

time of their death (mortalities were monitored hourly) or following twelve hours KMnO4

exposure. Fish that survived the treatment were euthanized with buffered (sodium

bicarbonate) tricaine methanesulfonate (1 g/L dose) (Finquel, Argent Laboratories,

Redmond, WA) before fixation. Whole fish were fixed in 10% neutral buffered formalin.









After 48 hours of fixation whole fish were separated into two histology cassettes; one

cassette contained complete, whole gill sections and the other cassette contained three

sections of body tissue. After the gills were removed the sample was cut into three

transverse sections (or steaks) cuts were made at the posterior eye, just cranially to the

pectoral fin and at the anus. Tissue was processed according to the schedule listed in

Appendix A.

Results

One hundred percent mortality was observed in all tanks at all salinities (2, 15, and

30 g/L) treated with 6 mg/L KMnO4. All fish from the 30 g/L salinity water treated with

6.0 mg/L KMnO4 died by the third hour of observation, while the 2 g/L and 15 g/L

salinity group fish treated with 6.0 mg/L KMnO4 all died by the sixth hour of

observation. Mortality was not observed in the three other tanks treated with 2 mg/L

KMnO4. Behavior observations were recorded and it was determined that fish generally

exhibited the following behaviors in sequential/chronological progression 1) increased

opercular movement; 2) erratic swimming; 3) intermittent loss of equilibrium or lying on

the bottom resting; and 4) the complete loss of equilibrium. Microscopic examination of

control tissue samples were within normal limits for the gills and other internal organs

(e.g. kidney, liver, spleen, intestine). All gill tissue samples collected from KMnO4

treated fish showed mucous cell infiltration and hyperplasia and inflammatory cell

infiltration and hyperplasia. The 15 g/L and 30 g/L salinity treatment at 6.0 mg/L

KMnO4 showed severe inflammation, expansion of the lamellar interstitium by edema

and inflammatory cells with lifting off of the epithelium, lamellar fusion and necrosis of

the gill epithelium. The gill samples offish from the 30 g/L salinity, 2 mg/L KMnO4

treatment were also beginning to show signs of epithelial lifting from edema and lamellar









fusion. The whole body sections of KMnO4 treated fish appeared to be within normal

limits for this species.

Discussion

The results of this pilot study suggest that KMnO4 caused more severe gill damage

to fish as the salinity increased. A more thorough, replicated experiment will test this

hypothesis. The fish in 30 g/L salinity group treated with 6.0 mg/L KMnO4 died faster

than those in the 2 and 15 g/L salinity treatments. These results suggest that fish in the

30 g/L salinity treatment group were more severely effected by the KMnO4 treatment. A

new experiment will be designed to test levels of KMnO4 that are not toxic to 100%

sailfin mollies (i.e., less than 6 mg/L KMnO4) to determine ifKMnO4 causes more severe

gill damage and possibly higher mortality at higher salinity levels.














CHAPTER 3
MATERIALS AND METHODS

Experimental Procedures

Experimental Fish and Quarantine Procedure

One thousand five hundred sailfin mollies were obtained from Segrest Farms in

Gibsonton, Florida. Upon arrival at the University of Florida Tropical Aquaculture

Laboratory (Ruskin, Florida) fish transport bags were placed in holding tanks for 30

minutes for temperature acclimation. Fish were exposed to a saltwater bath of 25 g/L for

approximately 5 minutes (Stoskopf 1993) to remove ectoparasites. All salt used in this

experiment was made with Instant Ocean salt mix (Aquarium Systems, Mentor, Ohio).

Fish were weighed in groups of approximately 100 (each individual bag with water) on

an electronic scale, netted out, and then placed into one of three holding tanks (680 L

each, 5 g/L salinity water). The bag and water were reweighed after fish were released to

determine an actual weight of fish in the bag. Fish were kept in the holding tanks until

experimental salinity acclimations (described below) were complete. Any sailfin mollies

that died during the salinity acclimation period were necropsied (including microscopic

gill, skin, and fin examination) to ensure that the sailfin mollies were remaining as free of

parasites as possible and for the early detection of other problems.

Fish in the holding tanks were acclimated to water salinities of 2 g/L, 15 g/L, or

30g/L, respectively. A reservoir tank containing no fish, but holding approximately 680L

of 30 g/L salinity water was used for water changes. The water in the reservoir tank was

made with Instant Ocean salt mix and well-water and was aerated for 24 hours prior to









water use (see Appendix B for typical composition). This water was added to the tanks

to adjust the salinities upward. Well water was added to decrease salinity. Salinities

were verified using a refractometer (Aquatic Eco-Systems, Apopka, Florida).

Fish were fed twice a day, five days a week a generic bulk tricolor flake food

(Zeigler Tri-color flake, Gardners, Pennsylvania). The weight of the total fish population

in each holding tank (about 500 fish) was 1247.5 grams (2 g/L salinity water), 1252.5

grams (15 g/L salinity water), and 1249.5 grams (30 g/L salinity water). The

approximate average weight per animal was 2.5 grams. Based on those weights, fish

were fed 12.5 grams (1% of total body weight in holding tank) of food twice per day, five

days a week.

Acclimation Procedure

Sailfin mollies were divided into one of three different holding tanks (500 fish per

tank) that were maintained at 5 g/L salinity. Freshwater was considered 2 g/L salinity

and the holding tank being adjusted to that salinity was not adjusted until the seventeenth

day of acclimation. The salinity of the other two holding tanks was raised twice a week

in increments of 5 g/L salinity.

During acclimation an active, air-driven, sponge biofilter was maintained at each of

the salinity levels that the fish would be held (10, 15, 20, 25, and 30 g/L) during the

acclimation period as a replacement or back-up filter. Filters were started 3 weeks before

the sailfin mollies arrived; the filters were fed daily with ammonia until they were needed

in the fish holding tanks. When the salinity was increased in the holding tanks, the "old"

biofilters were replaced with a "new" active biofilter that corresponded with the new

salinity of the holding tank. Seventeen days after fish arrival the desired salinities of 2

g/L, 15 g/L, and 30 g/L, respectively, were reached. Fish were maintained in the holding









tanks for five days more before being distributed to the treatment tanks. Salinity was

monitored daily using a refractometer throughout the acclimation period and salinity was

adjusted as needed.

After acclimation to the appropriate salinity group, the fish were divided into the

thirty-six, 75.7-L treatment tanks filled to 68 liters. Twelve tanks were randomly

assigned to each of the three salinities being tested (2, 15, and 30 g/L). Fish were

monitored in the 75.7-L tanks for three days before being exposed to KMnO4. Each tank

contained an air-driven sponge filter, which also provided aeration. Each tank was

stocked with 37 fish, an average biomass of 86.4 grams per tank (fish were reweighed

with an electronic scale prior to dispersal). Treatments were assigned to individual tanks

using the complete randomized block method (Ott RL and Longnecker M 2001). A

reservoir was available for each of the three salinity treatments so that water changes

could be done when needed.

Response Variables

Tanks were checked twice daily for dead fish and results were recorded.

Behavior was observed at the following times post KMnO4 treatment: time zero, 0.08,

0.16, 0.33, 0.66, 1.5, 3, 6, 12, 24, 48, 96, and 168 hours. Due to physical constraints,

observations for all 36 tanks could not all be made at the same time so a two-minute

delay was instated. Specifically in applying KMnO4, the first tank was dosed at 9:00

a.m., the second at 9:02 a.m., and so on until all tanks had been dosed in succession. Five

trained observers were used to score behavior of the experimental fish. Behavior scores

were categorized as follows:

1. within normal limits fish swimming normally in midwater,

2. slight increase in opercular movement,









3. marked increase in opercular movement, fish behavior mixed between resting and
actively swimming (usually erratically and repeatedly into the bottom and sides of
the tank),

4. beginning to lose the ability to maintain equilibrium or just laying on the bottom of
the tank, with obvious, labored respiration,

5. loss of equilibrium, floating throughout the tank with the current, very slow
opercular movement, fish on the verge of death.

Tissues for histologic processing were collected at three different time points: one

hour prior to KMnO4 treatment for controls, twelve hours after initiation of treatment,

and seven days post KMnO4 treatment exposure. Six whole fish per tank were taken for

each of the three histological sampling times. Prior to tissue collection, fish were

euthanized using a 1 g/L dose of buffered (sodium bicarbonate) tricaine methanesulfonate

(Finquel, Argent Laboratories, Redmond, WA) dissolved in water of corresponding

salinity, either 2, 15, or 30 g/L. Following euthanasia, a small opening was cut in the

fish's coelom and whole fish were submerged in 10% neutral buffered formalin for at

least 48 hours before processing.

Following fixation, all of the gill tissue was excised from each fish and placed in

individual cassettes for processing. The body of the fixed fish was also cut into three

transverse sections (or steaks, as in the pilot study Chapter 2) and placed in individual

cassettes for processing. All cassettes were then placed in decalcification solution (Cal-

EX, Fisher Scientific, Pittsburgh, PA) to remove any calcified material that could hinder

the microtomy of the tissue. The tissue was rinsed in tap water for four hours prior to

processing.

The tissues underwent a fourteen-hour tissue processing including alcohol (7

hours), xylene (3.5 hours), and paraffin wax (3.5 hours) see Appendix A for detailed

processing schedule. The tissue was embedded in paraffin. Tissue in the paraffin block









was cut at 5 micrometers and stained using a standard hematoxylin and eosin stain.

Slides were ready for viewing by light microscopy. Analysis consisted of scoring each

slide by scanning at 100X and examining in detail at 400X looking at approximately 50

gill filaments total using the following system:

1. Normal, includes background environmental damage (inflammation and mucous
cell infiltration) at the tip of the gill

2. Secondary lamellar damage including fusion; mucous cell and inflammatory cell
infiltration and hyperplasia, damage beginning to extend further from the tip of the
gill

3. Expansion of lamellar interstitium by edema and inflammatory cells with lifting off
of epithelium as well as fusion of lamellae

4. Necrosis and expansion of lamellar interstitium by edema and inflammatory cells in
50% or more of the lamellae

To limit observer bias the labels of each slide were covered with a slide label with

an arbitrarily assigned number. After all of the slides were scored and recorded, the

stickers were removed so that the scores could be matched with the slide identification.

Experimental Design

KMnO4 Treatment

The experimental design consisted of exposing sailfin mollies in each of the three

salinities (2, 15, and 30 g/L) to three different concentrations of KMnO4, and a control

group with no KMnO4 treatment (Table 3-1). The dosages listed in Table 3-1 are

calculated doses. Each treatment was replicated three times, making up 36 treatment

tanks.

Table 3-1. Experimental treatment combinations of KMnO4 concentrations and salinity
levels.
Salinity (g/L) KMnO4 Treatment (mg/L)
2 0, 0.5, 1.0, 3.0
15 0, 0.5, 1.0, 3.0
30 0, 0.5, 1.0, 3.0









Experimental Procedure

Salinity was monitored daily using a refractometer. Fish were not fed during the

experiment. During this time 25% water changes were done daily.

Potassium permanganate demand was determined for each salinity level using

water from randomly selected tanks one and two days prior to the KMnO4 treatment. The

resulting six numbers were used to calculate an average KMnO4 demand for all tanks at

the corresponding salinity (Table 3-2).

Table 3-2. Calculated KMnO4 demand for each salinity level. The mean KMnO4
demand was used to calculate the actual KMnO4 dosage for all treatments.
Salinity Level Mean KMnO4 demand
g/L mg/L
n=6
2 0.34
15 0.36
30 0.41

Three days after the fish had been transferred to the treatment tanks, water

chemistry parameters (salinity, pH, TAN, NO2) were tested in each tank. Water

chemistry was measured using a HACH Fish Farming kit (model FF-1A, HACH

Chemical Company, Loveland, CO) for the 2 g/L salinity tanks and a HACH Marine kit

(model FF-3, HACH Chemical Company, Loveland, CO) for the 15 and 30 g/L salinity

tanks. Water temperature was measured with a mercury thermometer. Water hardness

was only tested in make-up water; six 190-L vats of make-up water were made using

Instant Ocean saltmix and well water run through a reverse osmosis system (mixing was

done with aeration).

One hour before KMnO4 treatment, each sponge filter was removed and control

histological tissue samples were collected. Aeration was continued in each tank

throughout the KMnO4 treatment period. Observations were recorded for each replicate









at the following times: time zero, 0.08, 0.16, 0.33, 0.66, 1.5, 3, 6, 12, 24, 48, 96, and 168

hours. Following KMnO4 treatment (12 hours), a 50% water change was done on each

tank, followed by the addition of 5 mL of Kordon AmQuel (San Francisco, CA) to all

tanks (including control tanks) to deactivate the KMnO4 (as indicated by the AmQuel

label). Sponge filters were then replaced. Daily 25% water changes were done

thereafter.

Statistics

Descriptive statistics of water chemistry parameters were derived using Minitab

version 14.1 (State College, Pennsylvania). A one-way analysis of variance (ANOVA)

was performed on the nitrite and unionized ammonia values by the salinity level followed

by Tukey's HSD multiple comparison procedure. Mortality rate data was analyzed by

salinity and the KMnO4 concentration using a two-way ANOVA. The mortality rate data

was then analyzed by the salinity, KMnO4 concentration combination using one-way

ANOVA followed by Tukey's HSD multiple comparison procedure to find which

treatment concentrations were significant. An arcsin square root transformation of

percentage data was performed prior to analysis. Behavioral and histological scoring

data by the salinity, KMnO4 concentration combination were analyzed using Kruskal-

Wallis tests followed by Dunn's non-parametric multiple comparison test. Minitab

version 14.1 (State College, Pennsylvania) was used for all statistical analysis except

Dunn's multiple comparison test (Hollander and Wolfe 1973). A Type-I error rate (a) of

0.05 was used for all analyses except for Dunn's MCP. A Type-I error rate (a) of 0.15

was used, as recommended by Hollander and Wolfe (1973), for Dunn's MCP because of

the conservative nature of the test.














CHAPTER 4
RESULTS

Water Quality

Water chemistry parameters varied in each treatment tank. Water quality

measurements were taken prior to the KMnO4 application (Table 4-1). The nitrite levels

in the 15 g/L salinity treatment group were found to be significantly higher when

compared to the 2 and 30 g/L salinity treatment groups (F = 19.45, DF = 2, p < 0.001).

The unionized ammonia levels in the 2 g/L salinity treatment group were found to be

significantly higher when compared to the 15 and 30 g/L salinity treatment groups (F =

50.35, DF = 2, P < 0.001). Water hardness levels in the reservoir tanks were 256.6 mg/L

for 2 g/L salinity, 2,716 mg/L for 15 g/L salinity, and 5,480 mg/L for 30 g/L salinity (n =

1 for each salinity level). The temperature was 250C in all tanks (n = 36).

Table 4-1. Select water chemistry values for experimental tanks (n = 12) prior to KMnO4
treatments.
Salinity pH Unionized Nitrite (mg/L)
(g/L) Ammonia (mg/L)
2 8.33 / 0.0888 0.12 / 0.0435 0.14 / 0.0812
15 8.12/0.0389 0.02/ 0.01084 0.89/0.363
30 8.15 / 0.0522 0.03 / 0.01311 0.36 / 0.361
Data given in the format of mean / standard deviation.

Behavior

Fish behavior was affected by exposure to KMnO4. There were significant

differences among treatments (H = 30.48, DF = 11, P = 0.001) (Table 4-2). Fish that

were significantly impacted showed signs of distress including increased opercular









movement, erratic swimming, beginning loss of equilibrium, and total loss of

equilibrium.

Table 4-2. Kruskal-Wallis analysis of six-hour behavior scores by treatment
combinations (salinity KMnO4 concentration) (N = 3 tanks per treatment).
Treatment Combination: Median (see Average Rank Z
Salinity (g/L) KmnO4 footnote)
Concentration (mg/L)
2 -0.0 0 9.3 -1.57
2 -0.5 2 13.7 -0.83
2-1.0 3 20.0 0.26
2 -3.0 4 31.5 2.23
15 0.0 0 4.0 -2.49
15 0.5 3 16.3 -0.37
15- 1.0 3 23.8 0.92
15 3.0 4 31.5 2.23
30 0.0 0 5.7 -2.20
30 0.5 2 14.7 -0.66
30-1.0 3 20.0 0.26
30 3.0 4 31.5 2.23
H = 30.48 DF = 11 P = 0.001 (adjusted for ties)
Median key: 0 = within normal limits; 2 = marked increase in opercular movement,
erratic swimming; 3 = beginning loss of equilibrium, periods of rest on bottom of tank;
4 = total loss of equilibrium.


The Dunn's multiple comparison test demonstrated that the 2 g/L salinity, 3.0 mg/L

KMnO4 treatment (mean rank 27.5), the 15 g/L salinity, 3.0 mg/L KMnO4

treatment(mean rank = 27.5), and the 30 g/L salinity, 3.0 mg/L KMnO4 treatment (mean

rank = 27.5) were significantly different indicating that the sailfin mollies in those

treatment groups lost normal equilibrium and were highly effected by the KMnO4

treatment (Table 4-3). All other treatment groups did not show significance, indicated by

a critical value less than 26.3231. Behavior that was affected by exposure to KMnO4

treatment returned to normal within 24 hours after exposure, including fish exhibiting

extreme change.









Table 4-3. Dunn's multiple comparison test for behavior scores shows significant
treatment groups.
Treatment Combination: Salinity (g/L) + Difference in Mean Rank
KMnO4 Concentration (mg/L)
2 + 3.0 27.5
15 + 3.0 27.5
30 + 3.0 27.5
*A mean rank above the critical value (26.3231) indicates significance when compared to
the 15 g/L + 0.0 mg/L KMnO4 concentration (the least effected treatment or most
normal). The values were found using Dunn's multiple comparison test. All other
treatments did not show significance.


Histology

The slides of affected gill tissue showed secondary lamellar damage including

fusion and mucous and inflammatory cell infiltration and hyperplasia (Figure 4-1). More

severely affected gill tissue showed expansion of the lamellar interstitium by edema and

inflammatory cell infiltration with lifting off of the epithelium as well as secondary

lamellar fusion. The most severely affected gill tissue showed severe necrosis as well as

expansion of the lamellar interstitium by edema and inflammatory cells with lifting off of

the epithelium and secondary lamellar fusion.

There were significant differences among treatments (H=51.06, DF=11, P<0.001)

(Table 4-4). Significance in the 15 g/L salinity, 3.0 mg/L KMnO4 indicates expansion of

the lamellar interstitium by edema and inflammatory cells with lifting off of the

epithelium as well as secondary lamellar fusion (Figure 4-2). Significance in the 30 g/L

salinity, 3.0 mg/L KMnO4 treatment indicates necrosis of the gill tissue as well as

expansion of the lamellar interstitium by edema and inflammatory cells with lifting off of

the epithelium and secondary lamellar fusion (Figure 4-3). Slide scores of treated fish

returned to normal limits by the 168th hour.












Table 4-4. Kruskal-Wallis analysis of the slide score by the salinity/KMnO4 treatment
combinations.
Treatment Combination N= Median Average Rank Z
Salinity (g/L) KmnO4
Concentration (mg/L)
2-0 54 0 252.9 -2.66
2-0.5 54 1 268.9 -1.98
2-1.0 54 1 318.7 0.11
2-3.0 54 1 318.7 0.11
15-0 54 1 327.6 0.49
15-0.5 54 1 281.9 -1.44
15-1.0 54 1 348.1 1.37
15-3.0 54 1 391.9 3.20
30-0 54 0 240.5 -3.18
30-0.5 54 1 325.8 0.41
30-1.0 54 1 313.4 -0.11
30-3.0 36 2 446.3 4.41
H = 65.34 DF = 11 P < 0.001 (adjusted for ties)
Median key: 0 = within normal limits; 1 = secondary lamellar damage including fusion,
mucous and inflammatory cell infiltration and hyperplasia; 2 = secondary lamellar
damage including fusion, mucous and inflammatory cell infiltration and hyperplasia.
The 30 g/L salinity, 3.0 mg/L KMnO4 treatment groups shows n=36 due to mortality.
This table includes scores from all tissue collection times (zero, 12 hours, and 168 hours).

Table 4-5. Dunn's multiple comparison test for histology slide scores shows significantly
different treatments.
Treatment (g/L salinity Significantly Different Difference in Critical Value
mg/L KMnO4) From: (g/L salinity mg/L Mean Rank
KMnO4)
15- 3.0 2- 0.0 139.0 116.535
15- 3.0 2- 0.5 123.0 116.535
15- 3.0 30-0.0 151.4 116.535
30 3.0 2 0.0 193.4 130.290
30 3.0 2 0.5 177.4 130.290
30 3.0 15 0.5 164.4 130.290
30 3.0 30 0.0 205.8 130.290
30- 3.0 30- 1.0 132.9 130.290
A mean rank above the critical value (116.535) indicates significance when compared to
the treatment indicated. All other treatments did not show significance.

























U.4PE- -" U14 7-A1 Zr AU IM KI B
Figure 4-1. Gills from sailfin mollies in 2 g/L salinity water treated with KMnO4. A(200X, 0.0 mg/L
KMn04). Secondary lamellar structure is well-maintained (Slide Score = 0). B(200X, 3.0
mg/L KMnO4). Secondary lamellar fusion, as indicated by the arrow, is commonly seen in
this treatment group (Slide Score = 1). Stained by H&E.















A [
















Figure 4-2. Gills from sailfin mollies in 15 g/L salinity water treated with KMnO4. A (200X, 0.0 mg/L
KMnO4). The secondary lamellar structure is well-maintained (Slide score = 0). B(200X)
and C(400X) (3.0 mg/L KMnO4). There is expansion of the lamellar interstitium by edema
and inflammatory cells with lifting off of the epithelium as well as fusion of the lamellae
(Slide score = 2). Stained by H&E.








































Figure 4-3. Gills from sailfin mollies in 30 g/L salinity water treated with KMnO4. A.
(200X, 0.0 mg/L KMnO4). Secondary lamellar structure is well-maintained
(Slide Score = 0). B(200X) and C(400X) (3.0 mg/L KMnO4). Necrosis of the
lamellae, fusion of the secondary lamellae, as well as lifting of the epithelial
layer of cells by expansion of the lamellar interstitium by inflammation and
edema was common in this treatment group (Slide score = 3). Stained by
H&E.

Mortality

Salinity and KMnO4 concentration were found to have some significantly

different treatment groups (Table 4-6). Certain treatment combinations were also found

to be significantly different (Table 4-7). The 30 g/L salinity, 3.0 mg/L KMnO4 treatment

had significantly higher mortality than all other treatments, with 100-percent mortality

(Figure 4-7). The 15 g/L salinity, 3.0 mg/L KMnO4 treatment had significantly higher









mortality than the 15 g/L salinity, 0.0 and 0.5 mg/L KMnO4 treatments, with 36.33-

percent mortality (Figure 4-6). The 2 g/L salinity, 3.0 mg/L KMnO4 treatment was not

found to be significantly different from other treatments, but that treatment had 11.33-

percent mortality (Figure 4-5). Other treatment groups demonstrated very low, non-

significant mortality over the observation period (168 hours) (see Figures 4.4, 4.5, 4.6,

and 4.7).

Table 4-6. Two-way ANOVA of the total percentage mortality by the salinity and the
KMnO4 concentration.
Source Degrees of Sum of Mean F-test P-value
Freedom Squares Square
Salinity 2 3566.2 1783.08 12.57 < 0.001
KMnO4 3 15327.2 5109.06 36.02 < 0.001
Concentration
Interaction 6 9085.4 1514.23 10.68 < 0.001
Error 24 3404.0 141.83
Total 35 31382.8


Table 4-7. One-way ANOVA analysis of the total mortality percentage by the treatment
combinations.
Source Degrees of Sum of Mean F-test P-value
Freedom Squares Square
Treatment 11 6.6463 0.6042 18.62 < 0.001
Combinations
Error 24 0.7790 0.0325
Total 35 7.4253












100-



80-


60-



40-



20-


0-

KMn04 (mg/L)
Salinity (g/L)


0.0 0.5 1.0 3.0
2


0.0 0.5 1.0 3.0 0.0 0.5 1.0 3.0
15 30


Figure 4-4. Cumulative Total Percentage Mortality Over 7 days by Salinity (fresh 2 g/L,
brackish 15 g/L, and salt 30 g/L) and KMnO4 Concentration (0.0, 0.5, 1.0,
and 3.0 mg/L). Connected data points = mean.


100
90
S80
70
560 70 -0.0 mg/L KMnO(sub)4
50 __ -0.5 mg/L KMnO(sub)4
S-- A- 1.0 mg/L KMnO(sub)4
40- -3.0 mg/L KMnO(sub)4
R x 3.0 mg/L KMnO(sub)4


Time (hours)


Figure 4-5. The cumulative total percentage mortality shown over time for the 2 g/L
treatments.












100
90
> 80
S70
S60
50
0) -3
S40
2 30
.- 20
10
0


- 0.0 mg/L KMnO(sub)4
- 0.5 mg/L KMnO(sub)4
- A- 1.0 mg/L KMnO(sub)4
- 3.0 mg/L KMnO(sub)4


O 0z Nb bb


Time (hours)



Figure 4-6. Cumulative total percentage mortality shown over time for the 15 g/L
treatments.


- 0.0 mg/L KMnO(sub)4
- -- 0.5 mg/L KMnO(sub)4
- A- 1.0 mg/L KMnO(sub)4
- X- 3.0 mg/L KMnO(sub)4


0- 0t 10 0 0 N 'b\, O O O

Time (hours)



Figure 4-7. Cumulative total percentage mortality shown over time for the 30 g/L
treatments.


K K- -X X -x


100
90
S80
" 70
I 60
e 50
* 40
2 30
- 20
10
0


-At -A-A
I MA














CHAPTER 5
DISCUSSION AND CONCLUSIONS

Salinity/KMnO4 Treatments

Potassium permanganate use in freshwater aquaculture is common. The

concentration at which toxic effects are seen is affected by a variety of factors such as

water quality and species specific sensitivity. The effectiveness of KMnO4 is related to

the amount of oxidizable material in the water, i.e. organic and inorganic material, which

comprises the KMnO4 demand of the water (Tucker 1984). It has also been suggested

that water of high pH can cause manganese dioxide to precipitate (Stuart 1983 and Noga

1996). The purpose of this study was to determine if KMnO4 creates toxicity problems

for sailfin mollies held in saltwater compared to cohorts maintained in freshwater. This

study examined the effect of KMnO4 on the sailfin molly maintained at different salinity

levels and at different KMnO4 concentrations.

Tanks treated with 2 g/L salinity, 3.0 mg/L KMnO4 demonstrated 11.33%

cumulative mortality (Figure 4-4) throughout the entire 168-hour experiment.

Histological results in this experimental group showed only minimal secondary lamellar

fusion and mucous and inflammatory cell infiltration and hyperplasia in this same

treatment group (Figure 4-1). Sailfin mollies in the 2 g/L salinity water treated with 3.0

mg/L KMnO4 lost equilibrium in the water, demonstrating that they were negatively

affected by the KMnO4 treatment. The 2 g/L treatments also had a high mean ammonia

level. Mortality rate in this group may have been affected by the added stress from the

high ammonia levels in the water. The 3.0 mg/L KMnO4 concentration is higher than









that of the usual recommended dose of 2.0 mg/L KMnO4. It is possible that the 3.0 mg/L

KMnO4 dose is above the safety range of KMnO4 treatment for sailfin mollies kept in 2

g/L salinity water causing minimal mortality.

Tanks treated with 3 mg/L KMnO4 compared to control tanks treated with 0.0

mg/L KMnO4, were visually different due to KMnO4 concentration (the higher the

KMnO4 concentration the more purple the color of the water). This may cause observers

to approximate the KMnO4 concentration level, potentially creating bias. The behavior

scoring system was made in an attempt to allow the observer to judge the fish solely on

their behavior without factoring in the color of the water. The combined results of

behavior, mortality rate, and histological analysis suggests that KMnO4 treatment may

not be a safe application for the 2 g/L salinity, 3.0 mg/L KMnO4 even though it is not

significantly different from other treatments.

Use of KMnO4 in 15 g/L salinity water is a higher risk treatment than it would be in

2 g/L salinity water. The histology scoring system revealed expansion of the lamellar

interstitium by edema and inflammatory cells with lifting off of the epithelium as well as

fusion of the lamellae. This damage was consistently observed in this treatment group

(Figure 4-2). The lifting of the epithelium away from the blood vessels presumably

results in decreased efficiency of gas exchange and ammonia excretion, and therefore

increases the risk of mortality. As the epithelial layer of the lamellae is moved further

from the blood vessels more space is created and it is more difficult for gas (oxygen,

CO2) and other metabolites to move through the epithelium into the blood vessel and vice

versa. This effect will at least slow gas exchange and at severe levels may stop gas

exchange thus killing the fish.









Sailfin mollies in the 15 g/L salinity, 3.0 mg/L KMnO4 demonstrated 36.33%

mortality. Mortality variability was high (minimum = 0 %, maximum = 79 %),

indicating some inconsistency between populations housed in different tanks within the

same treatment group. This may indicate water chemistry problems (high nitrite levels in

the 15 g/L salinity treatments) or that there is variability in how individual fish react to

the chemical. A 3 mg/L KMnO4 dose is higher than the recommended dose possibly

contributing to toxicity. Further testing with increased replication would be necessary to

clarify why there was so much variation in mortality among fish populations in the 15

g/L salinity water, 3.0 mg/L KMnO4 treatment group.

As in the 2 g/L salinity, 3.0 mg/L KMnO4, the 15 g/L salinity, 3.0 mg/L KMnO4

treatment groups demonstrated loss of equilibrium during the treatment period. In

conjunction with high mortality and the gill trauma observed, this suggests that the 15

g/L salinity water, 3.0 mg/L KMnO4 treatment holds a higher risk for sailfin mollies.

KMnO4 should probably not be considered as a treatment choice in 15 g/L salinity water

without conducting a small-scale animal safety test on the species being considered.

The 30 g/L salinity, 3.0 mg/L KMnO4 treatment was not safe for sailfin mollies in

this experiment. As in the 2 and 15 g/L salinity water, 3.0 mg/L KMnO4 treatment

groups, the 30 g/L salinity water, 3.0 mg/L KMnO4 treatment group fish lost equilibrium

during the treatment. The maximum behavior score of 4 indicated a loss of equilibrium,

floating throughout the tank with the current with very slow opercular movement. Loss

of equilibrium at this point may indicate that the fish is directing all of its energy towards

respiration, letting equilibrium take second priority, or is so severely debilitated and has

lost the ability to maintain its equilibrium.









Histological changes to the gill in the 30 g/L salinity water, 3.0 mg/L KMnO4

treatment were severe and given the maximum histological score of 3. Lesions included

necrosis and expansion of lamellar interstitium by edema and inflammatory cells in 50%

or more of the lamellae. Gill damage observed in this group was much more severe than

that observed in other treatment groups. The response of the gill to KMnO4 treatment, as

described above, may have reduced gas exchange to the point of death by suffocation in

fish from this treatment group.

Mortality of sailfin mollies in the 30 g/L salinity water, 3.0 mg/L KMnO4 treatment

group was significantly different from all other treatment groups tested in this

experiment. One-hundred percent mortality occurred within 6 hours of exposure to 3.0

mg/L KMnO4 in all replicate groups. The behavior, histology, and mortality results from

this experiment indicate that KMnO4 is toxic to sailfin mollies from the 30 g/L salinity,

3.0 mg/L KMnO4 treatment.

Water Chemistry Parameters

Water chemistry analyses among treatment groups were highly variable. Salinity

change can cause biological filtration activity to decrease or even discontinue (Hovanec

and DeLong 1996). Although an attempt was made to maintain consistent water quality

by adding a conditioned biofilter to each of the different salinities encountered by the

fish, water quality was variable, especially at the lower salinities. The differences in the

nitrification process may have been due to the differences in salinity affecting the rates of

bacteria infiltration of the filter. However, the highest mean UIA levels were observed in

the 2 g/L salinity and the highest NO2 levels were observed in the 15 g/L salinity. This

may have contributed to mortality data collected. The stress of coping with the water









chemistry problems added to the caustic KMnO4 treatment may have caused mortality in

some fish creating variability in the results.

Although the 15 g/L salinity, 3.0 mg/L KMnO4 treatment group had 36.33%

mortality, the highest mortality was seen in the 30 g/L salinity, 3.0 mg/L KMnO4

treatment. The 30 g/L salinity treatment group exhibited the most normal water quality

(in reference to the UIA and NO2 levels), showing that mortality in this treatment group

was probably not related to UIA or NO2 levels.

In this experiment the mean pH was 8.33 for 2 g/L salinity, 8.12 for 15 g/L salinity,

and 8.15 for 30 g/L salinity, demonstrating low variability. Previous studies (Noga 1996,

Stuart 1983) suggested that the high pH of saltwater was responsible for mortality of fish

because manganese dioxide may precipitate onto the gills. In this study the lack of

variation in pH between treatment tanks does not support this hypothesis.

Ideally pH should have been measured electronically instead of colorimetrically.

The colorimetric tests lack precision, leading to increased variability. In the future more

comprehensive water quality analyses and other methods for parameter detection should

be used that do not involve colorimetric tests.

In this experiment only a calculated dose of KMnO4 was reported. The actual dose

of KMnO4 was not tested because the spectrophotometric methods available are not

reliable when testing water containing chloride or saltwater (Delfino, University of

Florida, personal communication). There was no feasible option to determine the actual

KMnO4 concentration in all three salinities tested (2, 15, and 30 g/L, respectively).

Technology or methodology should be developed to determine the actual dose of KMnO4

in water regardless of salinity.









Future Research

The objective of this experiment was to determine toxicity of KMnO4 (0.0, 0.5, 1.0,

and 3.0 mg/L) at varying salinity levels (2, 15, and 30 g/L respectively) using the sailfin

molly as a model. The study demonstrated that a concentration of 3.0 mg/L KMnO4 in

water of low organic content was not safe for the sailfin molly in 2, 15 or 30 g/L salinity

water as indicated by mean mortality rates of 11.33, 36.33 and 100 percent, respectively.

This data suggests that KMnO4 is not safe for sailfin mollies at concentrations of 3.0

mg/L. The data in this experiment also suggests that KMnO4 is a higher risk treatment in

water with salinity of > 15 g/L.

The cause of observed KMnO4 related mortality can be speculated to include an

unknown reaction between KMnO4 and the salinity, oxidation-reduction by-products that

are toxic, or attributes of the sailfin molly that may not be present in other fish. Many

elements other than sodium and chloride are present in saltwater. Potassium

permanganate may be interacting with one of these elements present in Instant Ocean salt

mix (Aquarium Systems, Mentor, Ohio) producing a new compound that causes a toxic

reaction in the fish. As indicated by Mallinckrodt Baker (2001), KMnO4 is incompatible

with bromides, iodides, ferrous salts, and arsenites, all of which are contained in Instant

Ocean salt mix (Aquarium Systems, Mentor, Ohio) in various forms (see Appendix B).

The consequences of these interactions are unknown in fish, but could be potential

hazards. This should be looked into further to rule out other chemical reactions that

would result in the fish being exposed to another compound other than KMnO4.

One example of a chemical interaction that could be taking place in saltwater

involves KMnO4 and bromide. KMnO4 causes an oxidation-reduction reaction, similar to

that of ozone (Camel and Bermond 1998). When ozone is used in saltwater systems it









can produce hypobromous acid, hypobromite ion, and then bromate through the oxidation

of bromide (Tango and Gagnon 2003). These compounds may be toxic to fish in the

water by way of a pH change. It is possible that the oxidation-reduction reaction

produced by KMnO4 could have similar byproducts that cause toxicity. In future

experiments, pH and oxidation-reduction potential should be measured throughout to

monitor change.

The sailfin molly used in this experiment is a euryhaline species that is able to

adapt to differing salinities well. The sailfin mollies' ability to adapt to fluctuating

salinities and ion concentrations may have affected the results of this experiment. The

addition of KMnO4 may change the ionic balance of the water compared to the blood of

the fish, impacting osmoregulation. Osmotic homeostasis must be maintained in the fish

to maintain good health. Sailfin mollies may be better suited to living in fresh to brackish

water environments as that is more of their natural distribution. It is possible that at

higher salinities even though the sailfin molly is able to survive, it is not able to flourish.

A sailfin molly in saltwater may be under more environmental stress. Therefore, when

challenged with a treatment such as KMnO4, the fish is less able to cope with the added

stress. Experiments should be completed to see if this possible cause of mortality and gill

damage is unique to KMnO4 treatment or if it encompasses other types of chemical

treatment or stressors as well.

Conclusions

Gill damage such as expansion of lamellar interstitium by edema and inflammatory

cells with lifting off of epithelium as well as fusion of lamellae and in severe cases

necrosis was seen in the 15 and 30 g/L salinity, 3.0 mg/L KMnO4 treatment groups.

Mortality was 11.33% in the 2 g/L salinity, 3.0 mg/L KMnO4 treatment group, 36.33%






38


in the 15 g/L salinity treatment group, 3.0 mg/L KMnO4 treatment group, and 100% in

the 30 g/L salinity, 3.0 mg/L KMnO4 treatment suggesting that KMnO4 at the highest

concentration tested was not safe for sailfin mollies in this experiment. Results also

suggest that as the salinity of the water increases, the toxicity of KMnO4 to sailfin mollies

also increases.














APPENDIX A
PROCESSING SCHEDULE FOR THE SHANDON EXCELSIOR AUTOMATIC
TISSUE PROCESSOR

Table A-1. Processing schedule for the shandon excelsior automatic tissue processor
Reagent Used: Time in hours:
Alcohol Ethyl 50-75% 1:00
Alcohol Ethyl 1:00
Alcohol Ethyl 1:00
Alcohol Ethyl 1:10
Alcohol Ethyl 1:10
Alcohol Ethyl 75-100% 1:30
Xylene 1:00
Xylene 1:15
Xylene 1:15
Wax Paraffin 1:00
Wax Paraffin 1:15
Wax Paraffin 1:15















APPENDIX B
TYPICAL COMPOSITION OF INSTANT OCEAN SALT


Table B-1. Typical composition of instant ocean salt


Chloride
Sodium
Sulfate
Magnesium
Potassium
Calcium
Carbonate/bicarbon
Bromide
Strontium
Boron
Fluoride
Lithium
Iodide
Barium
Iron
Manganese
Chromium
Cobalt
Copper
Nickel
Selenium
Vanadium
Zinc
Molybdenum
Aluminum
Lead
Arsenic
Cadmium
Nitrate
Phosphate


Solution at Approximate Salinity of 35ppt
Instant Ocean Seawater*
(ppm) (ppm)
19,290 19,353
10,780 10,781
2,660 2,712
1,320 1,284
420 399
400 412
ate 200 126
56 67
8.8 7.9
5.6 4.5
1.0 1.28
0.3 0.173
0.24 0.06
less than 0.04 0.014
less than 0.04 less than 0.001
less than 0.025 less than 0.001
less than 0.015 less than 0.001
less than 0.015 less than 0.001
less than 0.015 less than 0.001
less than 0.015 less than 0.001
less than 0.015 less than 0.001
less than 0.015 less than 0.002
less than 0.015 less than 0.001
less than 0.01 0.01
less than 0.006 less than 0.001
less than 0.005 less than 0.001
less than 0.004 0.002
less than 0.002 less than 0.001
None 1.8
None 0.2


* Data for seawater values taken from An Introduction to the Chemistry of the Sea.
1998. M.E.Q. Pilson















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BIOGRAPHICAL SKETCH

Emily N. Marecaux grew up in Ashland, Maine. After graduating from high school

she attended college at The University of Findlay in Findlay, Ohio. While at The

University of Findlay she pursued a Bachelor of Science degree majoring in biology, pre-

veterinary science, with a minor in chemistry. After graduating in May of 2002, Emily

came to The University of Florida to work on a Master of Science degree from the

Department of Fisheries and Aquatic Sciences with a focus on fish health. Emily has

accepted a position at the University of Arkansas at Pine Bluff as a fish health extension

associate.