*Corresponding author: niccole_wandelear@fws.gov Copepods are the most numerous of the parasitic crustaceans affecting a wide range of marine and freshwater finfish worldwide (Woo 2006). Species of the genus Salmincola are parasitic copepods of major concern in wild and cultured salmonids. The incidence and spread of Salmincola spp. can be exacerbated by degraded environmental conditions, e.g., increased water temperatures, high organic loading, low dissolved oxygen, and overcrowding. Such parasitic crustaceans often open portals of entry for secondary, opportunistic pathogens, thereby enhancing morbidity and mortality of fish (Bandilla et al. 2006). Fish heavily infested with Salmincola spp. will flash, jump, or rub along hard surfaces as they try to rid themselves of the parasites. Fish may become darker in color, produce excessive mucus, exhibit fatigue in flowing water, and go off feed. As a result, reduced growth and productivity of fish populations may occur.

*Corresponding author: niccole_wandelear@fws.gov Flavobacterium columnare (causative agent of columnaris disease) is a Gram-negative bacterium that is often associated with high levels of mortality in a variety of freshwater fish species. Columnaris disease is a chronic-to-acute, external or systemic disease, which can infect virtually any wild or cultured fish (Plumb 1999). The worldwide distribution of columnaris makes it one of the most important diseases affecting aquaculture today (Morrison et al. 1981; Post 1987; Wagner et al. 2002; Thomas-Jinu and Goodwin 2004). As with most bacterial diseases, maintaining proper environmental conditions and practicing good management procedures can reduce the severity and occurrence of disease outbreaks (Post 1987; Jeney and Jeney 1995). Although columnaris is primarily an external disease, it may become systemic with or without advanced skin and gill necrosis (Noga 2000). Historically, systemic infections have been treated with oral or parenteral (e.g., intravenous or intramuscular injection) antibiotic administration (Tripathi et al. 2003).

Chloramine-T (CLT) was recently approved for use in the United States by the U.S. Food and Drug Administration (FDA) to control mortality in selected freshwater-reared finfishes diagnosed with bacterial gill disease or external columnaris disease. In support of this approval, we conducted a study to determine if a target dose of 12 mg/L CLT could be delivered for 60 min via a "charged," flow-through treatment protocol. The study was conducted in two production-size, linear-design, plug flow raceways devoid of fish. Each raceway was dosed twice, resulting in four replicate trials (N = 4). During each trial, CLT was added under static conditions to establish a target concentration of 12 mg/L. Inflow water was then resumed, and additional CLT stock solution was metered into the raceway for the 60-min treatment period. Water samples were collected from a matrix of 27 sampling locations (3 positions along raceway length x 3 positions across raceway width x 3 depths) for colorimetric determination of CLT concentrations at 0 min (after charging but before resuming water inflow), 30 min, and 60 min. Chloramine-T doses delivered (data from all sampling locations and times pooled) did not vary from trial to trial. Median CLT doses delivered were almost always less than 12 mg/L; however, all had corresponding 95% confidence intervals within 9-15 mg/L. Overall, the results of our study demonstrated that the treatment method can be used to deliver a target dose of CLT for 60 min in production-size raceways in a manner that was found acceptable to the FDA.

The absence of a suitable sedative allowing treated fish to be released immediately after recovery constrains research and poses a risk to fish and those handling them. The U.S. Food and Drug Administration's reliance on multi-taxon datasets represents a major hurdle in the approval process. Experiments were conducted with twelve freshwater taxa to assess time to induction and recovery of fish sedated with different doses of AQUI-S 20E (10 % eugenol), Benzoak (20 % benzocaine), or MS-222 (99.5 % tricaine methanesulfonate) administered under various conditions. A retrospective analysis was conducted to determine whether sedative dose, water temperature, dissolved oxygen concentration, and fish length or weight contributed to variation in induction and recovery times. A subsequent experiment with eugenol was conducted to further assess time to sedation as a function of water temperature and sedative dose. Generally, higher doses and warmer temperatures were associated with faster inductions. Warmer temperatures were also associated with more rapid recoveries, however, high doses tended to delay recovery. Positive relationships linking estimated respiration rates and times to induction and recovery suggest the effects of temperature and body size on sedation timing may be a function of oxygen consumption. Collectively, our results demonstrated that the response of fish to chemical sedatives is primarily a function of sedative dose and water temperature, and, to a lesser extent, fish size and dissolved oxygen, not taxonomic classification. Accordingly, we suggest that as much information could be gained from a single taxon evaluated under different conditions as experiments involving multiple fishes. We recommend those establishing data requirements for fish drug approvals review these findings and consider alternative experimental designs as means of addressing regulatory requirements more efficiently and with greater rigor.

Columnaris (causative agent, Flavobacterium columnare) is a widespread fish disease of concern among fish culturists in the USA. If left untreated, an entire population of fish may become infected, and morbidity and mortality may reach high levels. In virtually all instances, columnaris outbreaks require intervention to prevent significant losses. A number of sanitizing agents, most notably chloramine-T (CLT) and hydrogen peroxide (HP), have been used to control mortality associated with a variety of bacterial pathogens causing external infections. However, the majority of trials conducted to demonstrate the effectiveness of these chemicals, thereby gaining U.S. Food and Drug Administration approval for their use in treating fish infected with columnaris, have been conducted on salmonids. Accordingly, we conducted seven experiments to evaluate the effectiveness of CLT or HP to control mortality associated with external columnaris in Florida Largemouth Bass Micropterus salmoides floridanus and Bluegill Lepomis macrochirus. Treatment with CLT or HP significantly reduced cumulative mortality in five of the seven experiments. Cumulative mortality was strongly correlated to pretreatment mortality in treated and control tanks in the five Largemouth Bass experiments, suggesting that intervention at later stages of columnaris progression may result in less favorable outcomes. Odds ratios calculated for individual experiments indicated varying degrees of success in controlling mortality; however, meta-analysis of all experiments indicated treatment with either CLT or HP significantly increased probability of survival, regardless of fish species or test article. These results demonstrate that both chemicals can be effective in controlling mortality associated with external columnaris in Largemouth Bass and Bluegills and that timely treatment of fish will likely result in lower overall mortality. Received December 7, 2012; accepted March 4, 2013

Aquaflor (florfenicol, 50% with/without) is a potent, broad-spectrum, antibacterial agent with bacteriostatic properties that are active against a variety of Gram-positive and Gram-negative bacteria. This product is approved by the U. S. Food and Drug Administration for use on several fish species to control mortality associated with a variety of diseases, including columnaris (causative agent, Flavobacterium columnare). Two independent experimental trials were separately conducted to evaluate the effectiveness of Aquaflor to control mortality associated with columnaris disease. Aquaflor was administered in feed at a targeted daily florfenicol dosage of 10mg/kg of body weight for 10 consecutive days. Test species were fingerling Florida Largemouth Bass Micropterus salmoides floridanus and Bluegill Lepomis macrochirus. In each trial, 8 or 10 test tanks (4 or 5 treated, 4 or 5 control) were stocked with either approximately 473 bass (mean length = 6.4mm; mean weight = 3.3g) or 100 Bluegills (length = 10.3; weight = 25.2g). At the end of the 14-d posttreatment periods, mean cumulative mortality of bass in treated tanks was 5.7% per tank, which was significantly less than that in control tanks (12.0%). Mean cumulative mortality of Bluegills in treated tanks was 19% per tank, which was significantly less than that in control tanks (38%). Analysis of treated feed samples at the start of each trial verified the initial targeted dose of florfenicol was within 11% of the target dose for both bass and Bluegills. Based on these results, we concluded that Aquaflor-medicated feed homogeneously mixed to provide florfenicol at a daily dose of10mg/kg of body weight fed for 10 d was effective in controlling mortality in bass and Bluegill fingerlings, exposed to columnaris disease. Received October 31, 2012; accepted March 5, 2013

As a synthetic androgen, 17-methyltestosterone (17MT) is frequently used to redirect the course of sex differentiation by exposing the undifferentiated gonad to a sufficient dosage. This hormone has been widely accepted as a safe and effective treatment for sex reversal in many fish species, and it is administered to larval tilapia (312 d old) for approximate to 28 consecutive days to produce populations of >90% males. This study assessed the safety of 17MT-treated feed when administered to larval Nile Tilapia Oreochromis niloticus at one, three, and five times (i.e., 1x, 3x, and 5x) the proposed dosage of 9mg 17MT/kg fish daily for 28 consecutive days. Despite elevated total ammonia nitrogen levels measured during the last 5 d of the study, environmental conditions were acceptable for rearing tilapia. Fish fed aggressively regardless of the concentration of 17MT in the feed, behavior was considered normal with no dose-related differences detected, and no mortality was observed in the 3x treatment group. Fish that were treated with five times the proposed therapeutic dosage had significant pathological changes. Based on the results of this study, the 17MT margin of safety extends to at least 3x (27mg 17MT/kg fish daily) the proposed dosage of 9mg 17MT/kg fish daily when administered in feed for 28 d to Nile Tilapia. Received October 8, 2012; accepted December 7, 2012

Abstract Salmincola spp. infestations can adversely affect freshwater-reared salmonids. Control methods tested to date have had limited success; consequently, we conducted a pilot field trial to evaluate SLICE (0.2% emamectin benzoate [EB])–medicated feed to reduce a natural infestation of S. californiensis in freshwater-reared rainbow trout Oncorhynchus mykiss. Before the trial started, 96 of 1,500 rainbow trout broodstock held in a flow-through raceway were impartially captured, sedated, uniquely tagged, and returned to the raceway. Pretreatment S. californiensis infestation prevalence and intensity were 97% and 10.4 ± 7.6 (mean ± SD) adult female parasites per fish, respectively. Treatment was administered at 50 μg EB·kg fish−1·d−1 for 7 d. By the end of the trial (43 d posttreatment), infestation prevalence and intensity had decreased to 32% and 1.6 ± 1.1 adult female parasites per fish, respectively. These results suggest that SLICE-medicated feed can be used to reduce natural infestations of S. cali...

We evaluated the efficacy of a Terramycin 200 for Fish (TM200; 44.09% oxytetracycline [OTC] dihydrate) treatment regimen proposed for fluorescent marking of the vertebrae of Rainbow Trout Oncorhynchus mykiss. Test fish weighed 36.6 +/- 1.9g (mean +/- SD) at the start of treatment. The TM200-treated feed was administered to six tanks of fish at a target dosage of 82.7mg OTC center dot kg fish1 center dot d1 for 10 d, while nontreated control feed was administered to three tanks of fish (20 fish/tank in each treatment group). After a 22-d posttreatment period, vertebrae were extracted from all fish and were examined for fluorescent marks with a dissecting scope and ultraviolet light. The vertebrae of all treated fish were marked, whereas vertebrae of control fish were not marked. Consequently, the probability of marking success in treated tanks was significantly different from that in control tanks. The 95% CI for the proportion of treated tanks containing one or more nonmarked fish was 0.00 to 0.39. Analysis of treated feed samples revealed that the actual OTC dose administered to treated tanks was 73.4 +/- 0.1mg OTC center dot kg fish1 center dot d1 (mean +/- SD; 89% of target). The results indicate that the target TM200 treatment regimen administered in this study is suitable for the marking of Rainbow Trout vertebrae. Received April 23, 2012; accepted July 16, 2012

Gyrodactylus salmonis is a monogenean ectoparasite that can infest a variety of captive-reared salmonid fishes. The physical damage inflicted during severe infestations can cause osmoregulatory disturbances and potentially render individuals more vulnerable to secondary pathogens. If not treated, G. salmonis infestations can reduce growth and survival in affected fish populations. Many chemical compounds have been used to treat Gyrodactylus infestations; however, little information has been published about the use of hydrogen peroxide (H2O2) for Gyrodactylus control. Consequently, we conducted a trial to evaluate the efficacy of H2O2 in reducing a natural infestation of G. salmonis in freshwater-reared, adult rainbow trout Oncorhynchus mykiss. Triplicate tanks of adult rainbow trout (20 fish per tank; length = 45.6 +/- 5.8 cm and weight = 1.3 +/- 0.4 kg, mean +/- SD) were exposed to a static bath of H2O2 at a target dosage of 50 mg/L or hatchery water (sham treatment) for 30 min/d on two alternate days. Treatment efficacy was assessed at 2 and 7 d posttreatment via light microscopy examination of skin scrapes (one per fish) taken from 10 fish per tank on each day. At 2 d posttreatment, the mean abundance of G. salmonis in the H2O2 treated group (0.1 +/- 0.3 G. salmonis individuals per skin scrape) was significantly different from that observed in the sham-treated group (34.4 +/- 43.2 individuals per skin scrape). Also, at 7 d posttreatment, the mean abundance of G. salmonis in the H2O2-treated group (0.1 +/- 0.3 individuals per skin scrape) was significantly different from that observed in the sham-treated group (38.5 +/- 77.4 individuals per skin scrape). The percent reduction in mean abundance (treated group compared with control group) was greater than 99% at both 2 and 7 d posttreatment. In conclusion, the H2O2 treatment regimen that we used significantly reduced a natural infestation of G. salmonis in freshwater-reared, adult rainbow trout.

Salmincola spp. are ectoparasites of major concern in wild and cultured salmonids. These parasites can cause respiratory distress and facilitate the entry of secondary pathogens. Of particular concern in the United States is S. californiensis, which can infest all Oncorhynchus spp. and is restricted largely to freshwater. Bath treatments with formalin and hydrogen peroxide have traditionally been used to control infestations of some parasitic copepods in cultured salmonids; however, these treatments can be difficult to apply, expensive, and stressful to fish and have not been shown to be effective against S. californiensis. A more effective and efficient treatment method needs to be developed. SLICE (0.2% emamectin benzoate [EB]) is a commercial in-feed treatment that has been shown to be effective for the control of sea lice infestations in seawater-reared farmed salmon and trout. We postulated that EB might also be efficacious for the control of parasitic copepods such as S. californiensis on freshwater-reared salmonids. Four trials were conducted to evaluate the effectiveness of SLICE-medicated feed administered at a target dosage of 50 mu g EB.kg fish(-1).d(-1) for seven consecutive days to control infestations of S. californiensis on freshwater-reared rainbow trout. At the end of each trial (after either a 30- or 42-d posttreatment period), copepod prevalence in treated tanks was substantially reduced compared with the pretrial prevalence recorded in the reference populations. Additionally, a significant difference was detected in mean abundance between treated and control groups, with a 79-96% reduction in mean abundance among fish offered the EB-medicated feed. Based on the results of these trials, it was concluded that SLICE was efficacious in reducing infestations of S. californiensis on freshwater-reared rainbow trout.

Chloramine-T (CLT) is a candidate for approval for use in U.S. aquaculture to control mortality in freshwater-reared salmonids caused by bacterial gill disease (causative agent, Flavobacterium branchiophilum). The proposed treatment regimen is to administer CLT at 12-20 mg/L in a static or flow-through bath for 60 min/d on three alternate or consecutive days. To estimate a CLT margin of safety, defined as the highest dosing regimen above the proposed maximum therapeutic regimen at which no adverse effects are observed, we conducted seven experiments with fry, fingerling, and juvenile rainbow trout Oncorhynchus mykiss that examined mortality and an eighth experiment that examined mortality, gross pathology, and histopathology after CLT exposure. In each experiment, triplicate groups of fish were exposed to a range of CLT concentrations representing 0, 1, 1.5, 2, 2.5, 3, 3.5, 4, or 5 x the highest proposed dose (20 mg/L) for 3 x the proposed treatment duration (60 min) on three alternate or consecutive days at 8 degrees C or 14 degrees C. The survival of fry and fingerlings was unaffected by exposure to CLT concentrations as high as 100 and 60 mg/L, respectively (survival = 97.3-100%). Although the survival of juvenile fish was unaffected by exposure to 20 mg/L, exposure to higher CLT concentrations significantly reduced survival (<= 10.0% at 100 mg/L). Across experiments, 92% of all mortalities occurred within 20 h of the first exposure to CLT. The histopathological changes of most concern were associated with gill tissues, but these were evident only in moribund fish exposed to doses of 60 mg/L or higher. Based on analysis of the survival data, the margin-of-safety estimates were approximately 100 mg/L for rainbow trout fry, at least 60 mg/L for fingerlings, and 50-60 mg/L for juveniles. Tissue responses to CLT at these concentrations were minor and did not warrant decreasing these estimates.

We conducted a field trial to evaluate the effectiveness of Aquaflor (50% florfenicol) for controlling mortality associated with Streptococcus iniae in freshwater-reared subadult sunshine bass (female white bass Morone chrysops X male striped bass M. saxatilis). Bacterial samples collected from moribund fish representing a reference population were presumptively identified microbiologically and were later confirmed to be S. iniae by biochemical characterization and polymerase chain reaction. The trial comprised a 1-d acclimation period, 10-d treatment period, and 14-d posttreatment period. During the treatment period, Aquaflor-medicated feed was administered to treated tanks (N = 3) at a target dose of 10 mg of florfenicol x kg of fish(-1) x d(-1), and nonmedicated feed was administered to control tanks (N = 3). At the end of the posttreatment period, mean (+/- SD) cumulative mortality in treated tanks (9 +/- 11%) was significantly (P = 0.040) less than that in control tanks (52 +/- 13%). Analysis of medicated feed samples revealed that treated tanks had received an actual dose of 8.3 mg florfenicol x kg fish(-1) x d(-1) (83% of target). No florfenicol was detected in control feed samples. Although the actual florfenicol dose administered to treated tanks was less than the target dose, the trial was accepted by the U.S. Food and Drug Administration Center for Veterinary Medicine as demonstrating the efficacy of Aquaflor to control mortality associated with S. iniae in cultured sunshine bass populations.

In 2009, we conducted a study to evaluate the efficacy of Terramycin® 200 for Fish (TM200; 44.1% active oxytetracycline dihydrate) administered in feed at a target dosage of 3.75 g/100 lbs fish/day for 10 days for the skeletal (fluorescent) marking of fingerling rainbow trout (Oncorhynchus mykiss). The in-life phase of the study was conducted indoors at a mean water temperature of 10.3 oC and comprised a 1-day acclimation period (no feed administered), 10-day treatment period (TM200-treated feed fed to six treated tanks; nontreated control feed fed to three control tanks), and 22-day post-treatment period (control feed administered to all tanks). At the end of the posttreatment period, all fish were collected and individually frozen. One month later, all fish were thawed, and two vertebrae were extracted from each fish. Each vertebra extracted was cleaned and then evaluated under ultraviolet light and a dissecting scope for the presence and quality of a fluorescent mark. All vertebrae extracted from TM200-treated fish (n = 120) had clearly visible marks, whereas no vertebrae extracted from control fish (n = 60) were marked. Consequently, in this study, TM200 administered in feed at a target dosage of 3.75 g OTC/100 lbs fish/d for 10 day was effective for the skeletal (fluorescent) marking of fingerling rainbow trout. Results will be used to support a U.S. approval of an expanded skeletal marking claim for TM200.

*Corresponding author: molly_bowman@fws.gov Furunculosis (causative agent, Aeromonas salmonicida) is one of the oldest known bacterial fish diseases and is generally considered a disease of salmonids (Plumb 1999). Mortality in affected freshwater-reared salmonid populations and resultant economic losses to producers can be substantial (Clark and Scott 1989). Hence, maintaining healthy rearing conditions, administering preventative vaccines, and administering antimicrobial treatments are strategies routinely used to prevent furunculosis outbreaks or minimize mortality when outbreaks occur (Inglis et al. 1991).

Bacterial gill disease (BGD), caused by Flavobacterium branchiophilum and other species of yellow-pigmented, filamentous bacteria, is a common and potentially catastrophic disease of hatchery (freshwater)-reared fish. Chloramine-T (Chl-T) is a biocide proven effective for controlling mortality in freshwater-reared fish diagnosed with BGD. However, Chl-T is not approved by the U.S. Food and Drug Administration for such use. To generate data in support of a U.S. approval, we evaluated the effectiveness of Chl-T (administered at 12 mg/L of static bath water for 60 min/d on three alternate days) to control mortality caused by BGD in freshwater-reared chum salmon Oncorhynchus keta, Apache trout O. gilae apache, and rainbow trout O. mykiss. For each species, the mean percent total mortality in Chl-T-treated tanks (N = 3) was significantly less than that in control tanks (N = 3): chum salmon = 8.9% versus 99.7%, Apache trout = 39.2% versus 97.9%, and rainbow trout = 5.7% versus 25.8%. Because the Chl-T treatment regimen was efficacious for each species, we conclude that our findings support the approval of Chl-T for use in the USA to control mortality in freshwater-reared salmonids diagnosed with BGD.

In July 2001, we conducted a study to determine whether a target concentration of chloramine-T (a waterborne chemical) could be achieved and maintained for 60 min in linear-design, plug-flow hatchery raceways (devoid of fish) via a "charged" flow-through treatment methodology. In each of four independent trials, a raceway was charged to achieve the target concentration by turning off the inflow water (creating a static bath) and manually mixing in a premeasured volume of chloramine-T stock solution. Water inflow was then turned on, and the target concentration was maintained by metering additional chloramine-T stock solution into the inflow water via a calibrated chicken-watering system. To help verify chloramine-T concentrations during treatment, we built an apparatus to rapidly collect many water samples from throughout a raceway. The apparatus comprised three fixed sampling stations, each of which was equipped with 9 water collection devices (i.e., nine 60-mL plastic syringes fitted with fixed-length "suction needles" made of rigid polyvinyl chloride pipe threaded with flexible vinyl tubing) and 9-11 plastic bottles for storing the collected samples. During each of the four 60-min trials, water samples were collected at elapsed times of 0, 30, and 60 min; thus, 12 sampling events were conducted during the study. During each sampling event, three people (working simultaneously but independently) collected a total of 29 water samples (27 for chloramine-T dose verification and 2 for quality control). The time for one person to collect 9-11 water samples (50-60 mL per sample) from one sampling station averaged 1.5 min (SD = 0.382; n = 36) and ranged from 0.9 to 2.5 min. The apparatus was inexpensive, easy to build and use, and portable; it ultimately helped us verify the spatial and temporal distribution of chloramine-T in linear-design, plug-flow hatchery raceways during 60-min charged flow-through treatments.

Anesthetics are physical or chemical agents that act on an animal by initially inducing a calming effect and subsequently inducing loss of equilibrium, mobility, consciousness, and reflex action (Summerfelt and Smith 1990). Anesthetics are commonly used in the culture of captive fish and in the handling of wild fish. As such, fish anesthetic research has been conducted on many compounds, e.g., carbonic acid, sodium bicarbonate, quinaldine, benzocaine, and 2phenoxyethanol. Virtually all fish culturists, biologists, and fisheries managers are familiar with two U.S. Food and Drug Administration (FDA) approved anesthetics: Tricaine-S and Finquel (i.e., MS-222). Although both types of MS-222 are very effective fish anesthetics, their use requires a 21-day withdrawal period before harvestable fish can be released or slaughtered. Such a lengthy withdrawal period greatly limits the scope of their use; therefore, fisheries professionals have long recognized that there is an important need for a zero-withdrawal fish anesthetic. This need has led to research on clove oil, a naturally derived compound that contains 90 95% eugenol. Although clove oil is effective and inexpensive to use, it is a crude product, does not have a sponsor, and therefore stands no chance of gaining FDA-approval. A U. S. Department of Health and Human Services guidance document (Guidance for Industry Document 150) describes FDA’s current position on clove oil, that it is not legal to use. An alternative product, AQUI-S, has recently emerged as a compound that has a good chance of gaining FDA-approval as a zerowithdrawal anesthetic. The U.S. Fish and Wildlife Service’s (FWS) Aquatic Animal Drug Approval Partnership (AADAP) program has assumed primary responsibility for generating data to support such an approval for the use of AQUI-S on all fish species.