Use of hexaflumuron to control parasitic infections in farmed and wild fish

Abstract

The present invention describes a process for preparing a cold premix hexaflumuron solution in water, preferably sea water, with a salinity of about 3.5% to about 18%, preferably from about 3.5% to about 14%; and at a temperature of about 0ºC to about 12ºC, preferably at about 4°C, and wherein the cold premix solution is then further added to sea water with fish for the treatment of the fish against sea lice.

Description

[0001] USE OF HEXAFLUMURON TO CONTROL PARASITIC INFECTIONS IN FARMED AND WILD FISH

[0002] Field of the Invention

[0003] The present invention provides for a novel approach to preparing a hexaflumuron premix to be added to sea water for the treatment of fish against ectoparasitic infection from sea lice.

[0004] Background of the Invention

[0005] Parasitic infestations constitute considerable problems in the fish farming industry as well as in wild fish. Damages due to parasitic infestations result in considerable losses and increased workloads for the fish farmers. Infestation with sea lice (Lepeophtheirus salmonis, Caligus elongatus and C. rogercresseyi) is considered to be one of the most important disease problems in the farming of salmonids, especially in Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss). In addition to the costs that are associated with treatment, lower classification ratings of slaughtered fish and reduced growth rate due to reduced feed intake contribute to the economic losses for the fish farmer. In addition to the damages caused in farmed fish, research has shown that sea lice could be the most important single cause leading to weakening of several wild salmon stocks. The emigration of salmon smolt from river systems is often coincidental with rising seawater temperatures in the fjord and coastal areas, which leads to a massive attack of copepodites (sea lice larvae) on the smolt. An attack of 40 or more sea lice on salmon smolt is fatal to fish weighing less than 25 grams, and in several regions in Norway premature return of post-smolt sea trout has been observed. Up until now, the most common treatment of fish parasites involves bathing or immersing the fish in a treatment solution comprising an anti-parasitic compound. This includes both skin and gill parasites. Bathing in formalin is a widespread treatment against many parasites, especially in fresh water, while bathing in organophosphates (e.g., metrifonate, dichlorvos, azamethiphos), pyrethroids (e.g., pyrethrum, cypermethrin, deltamethrin) or hydrogen peroxide are the most common bath treatments against sea lice (e.g., L. salmonis and C. elongatus). These substances act directly on the parasites via the water, and a possible absorption of the active substance into the fish itself is unimportant for the effect of the active substances on the parasite. Other methods of treating fish against ectoparasites includes dietary feed admixture and peritoneal injection.

[0006] Hexaflumuron is a chitin synthesis inhibitor that is suitable for modern aquaculture treatment of fish against ectoparasites, particularly sea lice. WO1 999 / 063824 discloses aqueous preparations of hexaflumuron. Hexaflumuron is supplied as a crystalline solid or powder. Hexaflumuron solubility in organic solvents like dimethylsulfoxide (DMSO) and dimethylformamide (DMF) is about 10 mg / mL and is sparingly soluble in aqueous buffers. In water, it is practically insoluble (0.027mg / L) in water (18°C). Thus, for use in water, hexaflumuron is prepared as a concentrate in an an organic solvent like N-methyl-2-pyrrolidone (NMP). The drug product, Alpha Flux®, is a concentrated (100mg / mL) solution of hexaflumuron in NMP. This concentrated NMP solution is diluted in water to prepare an intermediate (1 g / L) solution (premix) which is then diluted in larger volumes of water with fish to be treated for a final hexaflumuron concentration of 2mg / L (2ppm). When admixed with water, hexaflumuron slowly precipitates out of the solvent and forms a cloudy solution thereby limiting the amount of dissolved hexaflumuron available for fish treatment against sea lice. Applicant has surprisingly found that the temperature and the salinity of the water to be used to prepare the intermediate (premix) solution dramatically effects the rate of precipitation thereby ensuring effective amounts of hexaflumuron for treating fish which invariably provides a longer duration of efficacy and potentially reduces the potential for resistance against hexaflumuron.

[0007] Summary of the Invention

[0008] 1 ) The present invention describes a process for preparing an intermediate (premix) solution of hexaflumuron from a concentrated organic solution to ensure greater concentrations of hexaflumuron for treating fish against ectoparasites, particularly sea lice. In one aspect, the concentrated solution of hexaflumuron is a concentrate containing about 100 mg / mL of hexaflumuron in an organic solvent. In another aspect, the organic solvent is N-methyl-2-pyrrolidone (NMP). In one aspect, the concentrated organic stock solution of hexaflumuron is obtained from ALPHA FLUX® sold by Pharmaq (Norway). The hexaflumuron is used as a bath immersion treatment for fish against sea lice. Fish are salmonids, sea bass, turbot, halibut, sea bream, kingfish, barramundi and grouper. In another aspect, fish are salmonids. In another aspect, the salmonids are Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss). Preferably, fish are Atlantic salmon. Sea lice are Lepeophtheirus salmonis, Caligus elongatus and Caligus rogercresseyi. Preferably, sea lice are Lepeophtheirus salmonis or Caligus elongatus.

[0009] In one aspect, is a process for diluting a concentrated organic solution of hexaflumuron in cold water that is about 0°C to about 12°C to prepare an intermediate (premix) hexaflumuron solution containing about 500mg / L to about 2000mg / L hexaflumuron, and wherein the premix is then added to a larger volume of water to prepare a final water concentration of about 2mg / L (2ppm) for treating fish against sea lice. In another aspect, is a process for diluting a concentrated organic solution of hexaflumuron with a concentration of about 10Omg / mL in cold water that is about 0°C to about 12°C to prepare an intermediate (premix) hexaflumuron solution containing about 500 to about 2000mg / L hexaflumuron, and wherein the premix is added to a larger volume of water to prepare a final water concentration of about 2mg / L (2ppm) for treating fish against sea lice. In another aspect, is a process for diluting a concentrated organic solution of hexaflumuron with a concentration of about 100mg / mL in cold water that is about 0°C to about 12°C to prepare an intermediate (premix) hexaflumuron solution containing about 500 to about 2000mg / L hexaflumuron, and wherein the premix is added to a larger volume of water to prepare a final water concentration of about 2mg / L (2ppm) for treating fish against sea lice. In another aspect, is a process for diluting a concentrated organic solution of hexaflumuron with a concentration of about 100mg / mL in cold water that is about 0°C to about 12°C and with about 3.5% to about 18% salinity to prepare an intermediate (premix) hexaflumuron solution containing about 500 to about 2000mg / L hexaflumuron, and wherein the premix is added to a larger volume of water to prepare a final water concentration of about 2mg / L (2ppm) for treating fish against sea lice. In another aspect, is a process for diluting a concentrated organic solution of hexaflumuron with a concentration of about 100mg / mL in cold water that is about 0°C to about 12°C and with about 3.5% to about 14% salinity to prepare an intermediate (premix) hexaflumuron solution containing about 500 to about 2000mg / L hexaflumuron, and wherein the premix is added to a larger volume of water to prepare a final water concentration of about 2mg / L (2ppm) for treating fish against sea lice. In another aspect, is a process for diluting a concentrated organic solution of hexaflumuron with a concentration of about 10Omg / mL in cold water that is about 4°C to about 12°C and with about 3.5% to about 14% salinity to prepare an intermediate (premix) hexaflumuron solution containing about 500 to about 2000mg / L hexaflumuron, and wherein the premix is added to a larger volume of water to prepare a final water concentration of about 2mg / L (2ppm) for treating fish against sea lice. In another aspect, is a process for diluting a concentrated organic solution of hexaflumuron with a concentration of about 100mg / mL in cold water that is about 4°C to about 8°C and with about 3.5% to about 14% salinity to prepare an intermediate (premix) hexaflumuron solution containing about 500 to about 2000mg / L hexaflumuron, and wherein the premix is added to a larger volume of water to prepare a final water concentration of about 2mg / L (2ppm) for treating fish against sea lice. In another aspect, is a process for diluting a concentrated organic solution of hexaflumuron with a concentration of about 100mg / mL in cold water that is about 0°C with about 3.5% to about 14% salinity to prepare an intermediate (premix) hexaflumuron solution containing about 500 to about 2000mg / L hexaflumuron, and wherein the premix is added to a larger volume of water to prepare a final water concentration of about 2mg / L (2ppm) for treating fish against sea lice. In another aspect, is a process for diluting a concentrated organic solution of hexaflumuron with a concentration of about 10Omg / mL in cold water that is about 4°C and with about 3.5% to about 14% salinity to prepare an intermediate (premix) hexaflumuron solution containing about 500 to about 2000mg / L hexaflumuron, and wherein the premix is added to a larger volume of water to prepare a final water concentration of about 2mg / L (2ppm) for treating fish against sea lice. In another aspect, is a process for diluting a concentrated organic solution of hexaflumuron with a concentration of about 100mg / mL in cold water that is about 12°C and with about 3.5% to about 14% salinity to prepare an intermediate (premix) hexaflumuron solution containing about 500 to about 2000mg / L hexaflumuron, and wherein the premix is added to a larger volume of water to prepare a final water concentration of about 2mg / L (2ppm) for treating fish against sea lice. In another aspect, is a process for diluting a concentrated organic solution of hexaflumuron with a concentration of about 100mg / mL in cold water that is about 4°C and with about 3.5% salinity to prepare an intermediate (premix) hexaflumuron solution containing about 500 to about 2000mg / L hexaflumuron, and wherein the premix is added to a larger volume of water to prepare a final water concentration of about 2mg / L (2ppm) for treating fish against sea lice. In another aspect, is a process for diluting a concentrated organic solution of hexaflumuron with a concentration of about 100mg / mL in cold water that is about 4°C and with about 8% salinity to prepare an intermediate (premix) hexaflumuron solution containing about 500 to about 2000mg / L hexaflumuron, and wherein the premix is added to a larger volume of water to prepare a final water concentration of about 2mg / L (2ppm) for treating fish against sea lice. In another aspect, is a process for diluting a concentrated organic solution of hexaflumuron with a concentration of about 100mg / mL in cold water that is about 4°C and with about 14% salinity to prepare an intermediate (premix) hexaflumuron solution containing about 500 to about 2000mg / L hexaflumuron, and wherein the premix is added to a larger volume of water to prepare a final water concentration of about 2mg / L (2ppm) for treating fish against sea lice. In another aspect, is a process for diluting a concentrated organic solution of hexaflumuron with a concentration of about 100mg / mL in cold water that is about 0°C to about 12°C to prepare an intermediate (premix) hexaflumuron solution containing about 500 to about 2000mg / L hexaflumuron, and wherein the premix is added to a larger volume of water at least 3 minutes after preparing the premix to prepare a final water concentration of about 2mg / L (2ppm) for treating fish against sea lice. In another aspect, is a process for diluting a concentrated organic solution of hexaflumuron with a concentration of about 100mg / mL in cold water that is about 0°C to about 12°C to prepare an intermediate (premix) hexaflumuron solution containing about 500 to about 2000mg / L hexaflumuron, and wherein the premix is added to a larger volume of water at least 5 minutes after preparing the premix to prepare a final water concentration of about 2mg / L (2ppm) for treating fish against sea lice. In another aspect, is a process for diluting a concentrated organic solution of hexaflumuron with a concentration of about 100mg / mL in cold water that is about 0°C to prepare an intermediate (premix) hexaflumuron solution containing about 500 to about 2000mg / L hexaflumuron, and wherein the premix is added to a larger volume of water at least 5 minutes after preparing the premix to prepare a final water concentration of about 2mg / L (2ppm) for treating fish against sea lice. In another aspect, is a process for diluting a concentrated organic solution of hexaflumuron with a concentration of about 100mg / mL in cold water that is about 4°C to prepare an intermediate (premix) hexaflumuron solution containing about 500 to about 2000mg / L hexaflumuron, and wherein the premix is added to a larger volume of water at least 5 minutes after preparing the premix to prepare a final water concentration of about 2mg / L (2ppm) for treating fish against sea lice. In another aspect, is a process for diluting a concentrated organic solution of hexaflumuron with a concentration of about 100mg / mL in cold water that is about 0°C to about 12°C and with about 3.5% to about 14% salinity to prepare an intermediate (premix) hexaflumuron solution containing about 500 to about 2000mg / L hexaflumuron, and wherein the premix is added to a larger volume of water at least 3 minutes after preparing the premix to prepare a final water concentration of about 2mg / L (2ppm) for treating fish against sea lice. In another aspect, is a process for diluting a concentrated organic solution of hexaflumuron with a concentration of about 100mg / mL in cold water that is about 0°C to about 12°C and with about 3.5% to about 14% salinity to prepare an intermediate (premix) hexaflumuron solution containing about 500 to about 2000mg / L hexaflumuron, and wherein the premix is added to a larger volume of water at least 5 minutes after preparing the premix to prepare a final water concentration of about 2mg / L (2ppm) for treating fish against sea lice. In another aspect, is a process for diluting a concentrated organic solution of hexaflumuron with a concentration of about 10Omg / mL in cold water that is about 0°C and with about 3.5% to about 14% salinity to prepare an intermediate (premix) hexaflumuron solution containing about 500 to about 2000mg / L hexaflumuron, and wherein the premix is added to a larger volume of water at least 5 minutes after preparing the premix to prepare a final water concentration of about 2mg / L (2ppm) for treating fish against sea lice. In another aspect, is a process for diluting a concentrated organic solution of hexaflumuron with a concentration of about 100mg / mL in cold water that is about 4°C and with about 3.5% to about 14% salinity to prepare an intermediate (premix) hexaflumuron solution containing about 500 to about 2000mg / L hexaflumuron, and wherein the premix is added to a larger volume of water at least 5 minutes after preparing the premix to prepare a final water concentration of about 2mg / L (2ppm) for treating fish against sea lice. In another aspect, is a process for diluting a concentrated organic solution of hexaflumuron with a concentration of about 100mg / mL in cold water that is about 4°C and with about 3.5% salinity to prepare an intermediate (premix) hexaflumuron solution containing about 500 to about 2000mg / L hexaflumuron, and wherein the premix is added to a larger volume of water at least 5 minutes after preparing the premix to prepare a final water concentration of about 2mg / L (2ppm) for treating fish against sea lice. In another aspect, is a process for diluting a concentrated organic solution of hexaflumuron with a concentration of about 100mg / mL in cold water that is about 4°C and with about 14% salinity to prepare an intermediate (premix) hexaflumuron solution containing about 500 to about 2000mg / L hexaflumuron, and wherein the premix is added to a larger volume of water at least 5 minutes after preparing the premix to prepare a final water concentration of about 2mg / L (2ppm) for treating fish against sea lice.

[0010] Description

[0011] Figures:

[0012] Figure 1 . Hexaflumuron Premix Absorption at RT, 12°C and 4°C

[0013] Figure 2. Hexaflumuron Dilutions at 4°C

[0014] Figure 3. Hexaflumuron Dilutions at 12°C

[0015] Figure 4. Hexaflumuron Absorbance in Fresh Water and Sea Water

[0016] Figure 5. Hexaflumuron Absorbance at Different Salt Concentrations at 4°C Figure 6. Hexaflumuron Absorbance at Different Salt Concentrations at 12°C Definitions

[0017] As used herein, the term “about”, refers to the indicated value of the variable and to all values of the variable that are within the experimental error of the indicated value (e.g., within the 95% confidence interval for the mean) or within 10 percent of the indicated value, whichever is greater. As such, since the temperature gradient of 0°C to 12°C, is a tight gradient, a thermometer reading, for example, of 3.5°, 3.6°, 3.7°, 3.8°, 3.9°, 4.1°, 4.2°, 4.3°, and 4.4° can be construed as about 4°C and is contemplated to also be 4°C. Similar temperature values are contemplated for each temperature point within the claimed range.

[0018] “Treatment", "treating", and the like, as used herein, unless otherwise indicated, refers to reversing, alleviating, or inhibiting the parasitic infection, infestation, or condition. As used herein, these terms also encompass, depending on the condition of the fish, preventing the onset of a disorder or condition, or of symptoms associated with a disorder or condition, including reducing the severity of a disorder or condition or symptoms associated therewith prior to affliction with said infection or infestation. Thus, treatment can refer to administration of the hexaflumuron to a fish that is not at the time of administration afflicted with the infection or infestation. Treating also encompasses preventing the recurrence of an infection or infestation or of symptoms associated therewith.

[0019] “Water”, as used herein, unless otherwise indicated, refers to water that is fresh water, for example water from a tap or natural source that generally has a salinity value less than about 0.5 parts per trillion (ppt); sea water that can be obtained directly from the sea or ocean that has a salinity value of about 31 ppt to about 38ppt, and brackish water with a salinity value of about 0.5 ppt to about 30ppt. Water is also defined as water with added sodium chloride to increase the salinity than hat which was determined for the water sample depending upon its source. For example, hypertonic salinity is herein defined as water with a salinity >3.8% ( / .e. , >38ppt).

[0020] “Vessel”, as used herein, unless otherwise indicated, refers to a specialized tank that holds water and fish and is used for the treatment of said fish against sea lice by adding hexaflumuron to the water. Solubility is the ability of a substance, the solute, to form a solution with another substance, the solvent. In accordance with solubility and temperature of a solvent, it is generally understood that solubility of a solid increases as the temperature of the solvent increases; and that as the temperature decreases, solubility of the solid decreases. As the temperature of a solvent decreases, the solute precipitates out of the solvent solution. However, in the case of preparing an aqueous solution of hexaflumuron, it was noted that the hexaflumuron precipitated out of solution at greater levels when the solution was about 12°C than when the solution was colder, for example around 4°C. Treatment of fish against sea lice with hexaflumuron by immersion (bathing) in a solution of hexaflumuron has been performed over recent years. In essence, a concentrated organic solution of hexaflumuron containing about 100mg / mL has been diluted in water to prepare a premix intermediate solution. This premix is then further diluted in larger volumes of water containing fish at a concentration of about 2mg / L to treat the fish against sea lice. The water to prepare the premix is about 12°C, the same temperature of the water containing the fish. The average temperature of sea water containing fish to be treated for sea lice is considered to be about 12°C. When the premix is prepared, small micron size particles of hexaflumuron begin to precipitate out of the solution, which can be visually observed as a cloudy appearance. As such, the amount of hexaflumuron available for treating fish in a larger volume of water held in a well boat or a tarpaulin is relatively reduced. Thus, there was a need for improving the amount of available hexaflumuron in the water for treating fish against sea lice.

[0021] Ultraviolet-visible (UV-Vis) spectroscopy is a widely used technique to quantify the absorbance of light as it passes through a medium. As light passes through the test sample it is absorbed by precipitant which is then presented as a graph of absorbance, optical density or transmittance as a function of wavelength. The absorption spectrum maximum for hexaflumuron has an optical density of 600 nanometers. By measuring a specific concentration of hexaflumuron in the intermediate (premix) solution and fish treatment water with different concentrations of hexaflumuron, the amount of precipitation is quantified over time. As hexaflumuron precipitates in the aqueous solution, the solution becomes cloudy in appearance, thereby absorbing more light. This absorption is graphed against time to portray the amount of precipitation and level of drug available for the immersion treatment.

[0022] Although the vast majority of sea water has a salinity of between 31 and 38 g / kg ( / .e., 3.1-3.8%), sea water is not uniformly saline throughout the world. Sea water salinity is also expressed as a ratio of salt (grams) to a liter of water. There is typically close to 35grams of dissolved salts in each liter (35ppt), but ranges from 31 ppt to 38ppt, from 33ppt to 37ppt, and from 34ppt to 36ppt. As described herein, the salinity of sea water is considered to be, on average, 35ppt (3.5%). Further, sodium chloride can be added to water, regardless of its source, to prepare hypertonic solutions with a salinity >3.8%. Depending upon the amount of sodium chloride being added, these hypertonic solutions can have a salinity value that ranges from about >3.8% to about 36%, with a preferred range of about 7%, 14% 21 %, 28% and 36%. Intermittent hypertonic salinity solutions are also contemplated, for example, 4%, 6%, 10%, 15%, 18%, 23%, 26%, 30% 33%, and the like. A preferred intermittent hypertonic solution is about 14% salinity. These water and hypertonic solutions can then be used to prepare the premix hexaflumuron solutions for further dilution into sea water containing fish for treatment against sea lice.

[0023] Hexaflumuron is available for treatment of fish by immersion. The product is Alpha Flux® 100 and contains 100mg / mL hexaflumuron in an organic solvent, N-methyl- 2-pyrrolidone (NMP). An amount of the concentrate is diluted in water to attain an intermediate premix concentration of hexaflumuron at about 500mg / L to about 2000mg / L. The premix solution is diluted further by adding the premix to a larger volume of water containing fish to be treated against sea lice at a concentration of about 2mg / L of hexaflumuron. As described herein, this method of preparing the premix solution has had issues with precipitation of the hexaflumuron from the aqueous solution, thereby decreasing the available hexaflumuron available for treating fish. In an effort to modify this preparation regimen, Applicant unexpectedly found that by reducing the temperature of the water to about 4°C from 12°C before adding the concentrated hexaflumuron solution, they could reduce the amount of precipitate by 50% or greater thereby ensuring greater concentrations of hexaflumuron for fish treatment. Water is described as being fresh water, sea water and brackish water. The preferred water to be used for preparing the intermediate premix solution of hexaflumuron is sea water. Sea water is easily obtained by those preparing the premix solution prior to treating fish, particularly those on a well boat or for use with a tarpaulin. The water for the premix can be chilled by a number of different methods, including refrigeration and / or use of a water chiller. A water chiller removes heat from the process water and transfers it to surrounding air via a heat exchange process. Using a mechanical compressor and a refrigeration cycle, water chillers are able to cool the water to below ambient temperature. It is understood that untreated water freezes at 0°C (32°F). The refrigerant in the chiller's evaporator must be 10°F or more colder than the desired fluid set point to create heat transfer. Therefore, it is likely that the chiller may require a propylene glycol mixture to lower the fluid freeze point to chill the water to about 2°C to 12°C.

[0024] Experimental

[0025] In initial experiments, hexaflumuron absorbance was measured with a UV spectrometer every 2-minutes over time from a premix sea water solution (50mL) containing 1000mg / L hexaflumuron maintained at room temperature (RT, 21 -22°C), 12°C and 4°C. As shown in Figure 1 , hexaflumuron absorption increased at around 10 minutes, 19 minutes and 30 minutes for the samples at RT, 12°C and 4°C, respectively, showing the earlier increased amount of hexaflumuron precipitation in the RT sample.

[0026] In a subsequent experiment, it was shown that continual mixing of the premix samples with a magnetic stir bar over the duration of the study («40 minutes) versus no mixing in RT samples delayed precipitation by about 2-minutes (8 vs 10 minutes) with similar absorbance values thereafter. Similarly, mixing resulted in greater absorbance at about 15 minutes than non-mixed samples at 14-16°C. Mixing at 4°C increased the rate of precipitation which equilibrated at about 16 minutes wherein the non-mixed sample precipitation exceeded that seen from the mixed sample at around 24 minutes. Overall, the premix should be prepared at temperatures ranging from about 2°C to about 12°C and without continual mixing.

[0027] In another experiment, a premix solution of hexaflumuron (1000mg / L) was prepared at 4°C. Aliquots of the premix were added to separate sampling tubes at 4°C to prepare hexaflumuron concentrations at 250mg / L, 125mg / L and 62.5mg / L. Absorption was measured every two minutes. The premix precipitation was noticeable around 26-30 minutes while the serial dilution samples started showing signs of precipitation around 60 minutes for the 250mg / L sample and 90 minutes for the lower concentration samples. Figure 2. A similar experiment was conducted, but this time the premix was made at 12°C and diluted into sampling tubes with sea water also at 12°C. Absorption of the premix sample was noticeable around 20 minutes whereas the 250mg / L sample precipitation was noticeable around 38-40 minutes and the lower concentration sample absorption values were similar to those observed in the 4°C samples. Figure 3. Dilutions of 125mg / mL appeared to be the lowest concentration that was detectable by spectrophotometry, since there was no realized change in absorbance over time for the 62.5mg / L sample. Overall, precipitation of hexaflumuron for samples prepared at 4°C was minimized when compared to those prepared at 12°C.

[0028] In another study, premix (1 g / L hexaflumuron) was prepared in 4°C and 12°C sea water. At one minute and 5-minutes following preparation of the premix, samples were then diluted further in 12°C sea water at a concentration of 125mg / L to simulate tarpaulin treatment. Absorption was assessed at 60 minutes, 90 minutes and 120 minutes. Absorption was also measured at the same time points for a premix sample retained for 24 hours at 4°C before dilution to 125mg / L in the 12°C sea water. Absorption values are shown in Table 1 .

[0029] Table 1 . Absorption Values for Hexaflumuron at 4°C and 12°C

[0030] As can be observed from the Table 1 absorbance values, preparation of the premix in 4°C sea water prolongs the time before hexaflumuron precipitation when added to 12°C sea water. As such, when the premix hexaflumuron solution is prepared in 4°C sea water then added to the 12°C treatment sea water with fish, there is a greater concentration of hexaflumuron being administered to the treatment water to treat the fish against sea lice.

[0031] Overall, it was found that the premix solution of hexaflumuron should be prepared in cold sea water to minimize precipitation of the hexaflumuron prior to the premix being added to the 12°C sea water containing fish for treatment against sea lice.

[0032] In another study, hexaflumuron (1000mg / L) premix absorbance was measured in fresh water from the tap and sea water at 11 °C and 3.4°C. Hexaflumuron absorbance increased around 26 minutes for the water samples at 11 °C, around 28-30 minutes for fresh water at 3.4°C and between 30-36 minutes for sea water at 3.4°C. Figure 4. Colder water still increased the time to hexaflumuron precipitation.

[0033] In another study, hexaflumuron (1000mg / L) premix solution absorbance was measured at 4°C using fresh water, and fresh water spiked with sodium chloride (NaCI) to simulate the average salinity of sea water (3.5% salinity) and sea water at salt concentrations of 7% and 14% salinity. As can be observed in Table 2 and Figure 5, hexaflumuron precipitation, as measured by absorbance, was time dependent on the concentration of NaCI and / or salinity of the water.

[0034] Table 2. Absorption Values for Hexaflumuron (1000mg / L) at Different Salinity at 4°C.

[0035] In a follow-on study, cold 4°C premix (1 g / L) was prepared in fresh water, 3.5%, 7% and 14% salinity water which was then diluted into 12°C sea water for a final concentration of 125mg / L hexaflumuron. Absorption values were monitored starting at 40-minutes from dilution. Absorption results are shown in Table 3 and Figure 6. As can be observed, precipitation of hexaflumuron was delayed following dilution to 125mg / L into sea water (3.5% salinity) at 12°C when samples were prepared in 4°C water with salinity values of 3.5% to 14%, with the greatest delay seen for the 7% and 14% values, respectively.

[0036] Table 3. Absorption Values for Hexaflumuron (125mg / L) at Different Salinity at 12°C.

[0037] Overall, the studies indicate that hexaflumuron premix solutions made at colder temperatures results in slower precipitation of hexaflumuron based on absorbance values. Further, addition of sodium chloride to the water samples to achieve higher salinity values at 4°C also resulted in slower precipitation of hexaflumuron based on absorbance values, even when diluted to 125mg / L in 12°C sea water. Additionally, continual mixing of the premix samples resulted in faster precipitation.

[0038] In an in-vivo study, immersion of Atlantic salmon in treatment water containing 2ppm of hexaflumuron were assessed for hexaflumuron uptake. A premix of Alpha Flux® (100mg / mL) hexaflumuron was prepared in sea water containing 3.5% and 14% salinity at temperatures at 0°C, 4°C and 12°C. The premix was then added directly to separate 50L tubs each containing 15 fish (100g) either 1 -minute or 5-minutes after the premix was prepared. Fish were exposed to the hexaflumuron treatment for 2-hours. At 72-hours post treatment, tissue samples (filets (muscle / skin)) were obtained from all treatment groups and assessed for hexaflumuron uptake. As can be observed in Table 4, fish attained the highest concentration of hexaflumuron when the hexaflumuron was pre-mixed in 4°C sea water with 14% salinity when the premix was added to the treatment tub 5-minutes after preparation. Hexaflumuron filet concentrations were about 13% lower in fish at 4°C (3.5% salinity) and 0°C (14% salinity). The lowest concentrations were achieved when the premix water was at 12°C, and particularly at 3.5% salinity.

[0039] Table 4. Hexaflumuron Fish Concentrations

Claims

Claims1 ) A process for preparing a cold premix solution of hexaflumuron by diluting a stock solution of hexaflumuron in an organic solvent into a volume of cold water at a water temperature of about 0°C to about 12°C wherein the water is cooled by refrigeration or with the use of a water chiller.2) The process of claim 1 , wherein the stock solution of hexaflumuron is a 100mg / mL solution of hexaflumuron and wherein the organic solvent is N-methyl-2- pyrrolidone and the cold premix solution contains hexaflumuron at a concentration of about 500mg / L to about 2000mg / L.3) The process of claim 1 or 2, wherein the water has a salinity value of about 3.5% to about 14%.4) The process of claim 3, wherein the temperature of the cold premix solution is about 4°C.5) The process of claim 4, wherein the cold premix solution has a salinity value of about 3.5%.6) The process of claim 4, wherein the cold premix solution has a salinity value of about 14%.7) The process of any one of the preceding claims, wherein the cold premix solution is held for about 3 to about 7 minutes before further dilution to about 2mg / L into a treatment vessel with fish.8) A process for preparing a cold premix solution of hexaflumuron by diluting a stock solution of hexaflumuron in an organic solvent into a volume of cold sea water at a water temperature of about 0°C to about 12°C and wherein the sea water has a salinity value in the range of about 3.5% to about 14%.9) The process of claim 8, wherein the cold premix solution is about 0°C and has a salinity value of about 3.5%.10) The process of claim 9, wherein the cold premix solution has a salinity value of about 14%.11 ) The process of claim 8, wherein the cold premix solution is about 4°C and has a salinity value of about 3.5%.12) The process of claim 8, wherein the cold premix solution is about 4°C and has a salinity value of about 14%.13) The process of any one of the preceding claims, wherein the cold premix solution containing hexaflumuron is added to sea water containing fish for the treatment of sea lice on fish.14) The process of claim 13, wherein the fish are salmonids selected from Atlantic salmon Salmo salar) and rainbow trout (Oncorhynchus my kiss).15) The process of claim 13, wherein the sea lice are Lepeophtheirus salmonis, Caligus elongatus and Caligus rogercresseyi.

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