WO2023183442A1

WO2023183442A1 - Degradation of hexaflumuron in ozonated sea water

Info

Publication number: WO2023183442A1
Application number: PCT/US2023/015991
Authority: WO
Inventors: Marit Rode; Simen Johnsen OSTNAES; Yngve LYSTAD; Kathrin ELLESAT
Original assignee: Zoetis Services Llc
Priority date: 2022-03-24
Filing date: 2023-03-23
Publication date: 2023-09-28
Also published as: DK202370145A1

Abstract

The invention describes a process for degrading hexaflumuron from waste sea water treated with hexaflumuron that was used to delouse fish by adjusting the pH of the water to about 10 with a base; ozonating the water to achieve a total residual oxidant (TRO) level of about 4 to 9 mg/L; incubating the water after ozonation for a period of about 3 hours, or more; neutralizing the waste sea water after the incubation period by adding a reducing agent; and finally discharging the treated waste sea water back into the environment.

Description

DEGRADATION OF HEXAFLUMURON IN OZONATED SEA WATER

FIELD OF THE INVENTION

The present invention provides a new process for degrading hexaflumuron from waste sea water from aquaculture before discharging the water to the environment.

BACKGROUND OF THE INVENTION

Hexaflumuron, is a benzoylurea insect growth regulator that inhibits chitin synthesis in insect and acarid exoskeletons. Hexaflumuron is used for land-based insects like termites and for treating fish against sea lice.

Parasitic infestations constitute considerable challenges in the fish farming industry. This applies particularly to farmed fish in fresh and sea water, and preferably sea water. Infestation with sea lice (e.g., Lepeophtheirus salmonis, Caligus elongatus and C. rogercresseyi) is considered to be one of the most important disease problems in the farming of salmonids, especially 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 from louse scarring and reduced growth rate due to reduced feed intake contribute to the economic losses for the fish farmer.

A common method of treating fish against sea lice and other aquatic parasites is by bathing or immersing the fish in a treatment solution comprising a parasiticide. This includes both skin and gill parasites. Bathing fish in formalin has been a widespread treatment against many parasites particularly in fresh water; while bathing fish in organophosphates (e.g., dichlorvos and azamethiphos), pyrethroids (e.g., cypermethrin and deltamethrin), chitin synthesis inhibitors (e.g., hexaflumuron and diflubenzuron) or hydrogen peroxide are common antiparasitic immersion treatments.

Frozen and thawed fish processing has used ozone to sterilize, remove pesticide residues, antibiotics and hormones by soaking and rinsing the fish product(s) over a period of about 4-5 hours (Chinese Patent Publication No. 111165717A). Accordingly, the dissociated atomic oxygen (O) and hydroxyl groups (-OH) decompose organic matter, bacteria and microorganisms and can effectively degrade organic phosphorus, carbamates, pyrethroid residues and grease. According to Velioglu, et.al., “Effects of Ozone Treatment on the Degradation and Toxicity of Several Pesticides in Different Groups”, Journal of Agricultural Sciences, 24 (2018) pp. 245-255; some neonicotinoid, pyrethroid, methoxyacrylate, organophosphate and tetramic acid pesticides (e.g., thiacloprid, fenazaquin, azoxystrobin, chlorpyrifos, lambda cyhalothrin, spiromesifen and others) can be degraded with ozone in a citrate buffered distilled water sample (30 mL) at 15°C with ozone. Degradation rates ranged from 85-99%; however, some degradation products were shown to be more toxic than the parent pesticide, particularly against a susceptible non-target fresh water species (D. magna). These processes relied on ozone to remove pesticide residues from food products in controlled laboratory settings. Ozone is also used efficiently for industrial, domestic and drinking water purification. To date, use of ozone to degrade hexaflumuron from treated waste sea water following treatment of live fish with hexaflumuron prior to discharging the water into the environment has not been previously described.

On well boats, sea water and farmed fish are pumped into a large tank(s) on the boat. The sea water and fish are then treated with a parasiticide against ectoparasites. After treatment, the fish are transferred back to their holding pens, for example, large netted floating enclosures. The remaining treated waste sea water was routinely discharged into the environment where it was quickly diluted or sedimented. These discharges are regulated by the country of origin. These regulations and programs were developed as a means of preventing adverse impacts on the environment, including any detrimental activity against non-targeted organisms. Therefore, there is a need for reducing or eliminating hexaflumuron from the waste sea water after fish treatment before discharging the water into the environment.

SUMMARY OF THE INVENTION

In one aspect of the invention, is a process for degrading hexaflumuron from waste sea water containing hexaflumuron comprising: a) adjusting the pH of the water to a pH of about 10; b) adding ozone to the water to achieve a Total Residual Oxidants (TRO) level of about 4 to 9 mg/L (CI2); c) after ozonation, incubating the water for about 3 hours, or more; d) after incubation, neutralizing the water with a reducing agent; and e) discharging the neutralized water into the environment. In another aspect, the waste sea water pH is adjusted to a pH of about 10 with a base. In another aspect, the base is sodium hydroxide (NaOH) or potassium hydroxide (KOH). In another aspect, ozone is added to the waste sea water at a rate of about 1 to 10 mg/L/hour of ozone. In another aspect, the ozone is added to the waste sea water to achieve an amount of about 10 to 30 mg/L. In another aspect, the ozone is added to the waste sea water to achieve an amount of about 12 to 28 mg/L ozone. In another aspect, the ozone is added to the waste sea water to achieve an amount of 15 to 28 mg/L ozone. In another aspect, the ozone is added to the waste sea water to achieve an amount of about 15 to 22 mg/L ozone. In another aspect, the ozone is added to the waste sea water to achieve an amount of about 15 to 20 mg/L ozone. Since water quality, temperature and ozonation equipment varies, the needed output of ozone to be added to the water will also vary. In view of this variable, ozone is added to the waste sea water to achieve a TRO level of about 4 to 9 mg/L. In another aspect, the ozone is added to the waste sea water to achieve a TRO level of about 5 to 8 mg/L. In another aspect, the ozone is added to the waste sea water to achieve a TRO level of about 5 to 7 mg/L. In another aspect, the ozone is added to the waste sea water to achieve a TRO level of about 5 to 6 mg/L. In another aspect, the ozone is added to the waste sea water to achieve a TRO level of about 5 mg/L. In another aspect, the ozone is added to the waste sea water to achieve a TRO level of about 5.5 mg/L. In another aspect, the ozone is added to the waste sea water to achieve a TRO level of about 6 mg/L. In another aspect, after ozonation, the waste sea water is incubated for a period of about 3 hours, or more. In another aspect, the waste sea water is incubated for a period of about 3 to about 120 hours. In another aspect, the waste sea water is incubated for a period of about 3 to about 48 hours. In another aspect, the waste sea water is incubated for a period of about 3 to about 24 hours. In another aspect, the waste sea water is incubated for a period of about 3 to about 18 hours. In another aspect, the waste sea water is incubated for a period of about 3 to about 12 hours. In another aspect, the waste sea water is incubated for a period of about 3 to about 6 hours.

In another aspect, after incubation, the waste sea water is neutralized with a reducing agent. In another aspect, the reducing agent is selected from the group consisting of a sulfite or thiosulfate. In another aspect, the reducing agent is sodium sulfite, potassium sulfite, calcium sulfite, sodium thiosulfate, potassium thiosulfate or calcium thiosulfate. In another aspect, the reducing agent is sodium sulfite or sodium thiosulfate. In another aspect, the reducing agent is sodium sulfite. In another aspect, the reducing agent is sodium thiosulfate.

In another aspect, is a process for degrading hexaflumuron from waste sea water containing hexaflumuron comprising: a) adjusting the pH of the water to a pH of about 10 with NaOH or KOH, b) adding ozone to the water to achieve a TRO level of about 5 to 7 mg/L; c) after ozonation, incubating the water for a period of about 3 to 48 hours, d) neutralizing the water with a reducing agent selected from a sulfite or thiosulfate, and e) discharging the neutralized water into the environment. In another aspect, the sulfite is sodium sulfite, calcium sulfite or potassium sulfite, preferably sodium sulfite and the thiosulfate is sodium thiosulfate, calcium thiosulfate or potassium thiosulfate, preferably sodium thiosulfate.

In another aspect, is a process for degrading hexaflumuron from waste sea water containing hexaflumuron comprising: a) adjusting the pH of the water to a pH of about 10 with NaOH or KOH, b) adding ozone to the water to achieve a TRO level of about 5 to 7 mg/L; c) after ozonation, incubating the water for a period of about 3 to 24 hours, d) neutralizing the water with a reducing agent selected from a sulfite or thiosulfate, and e) discharging the neutralized water into the environment. In another aspect, the sulfite is sodium sulfite, calcium sulfite or potassium sulfite, preferably sodium sulfite and the thiosulfate is sodium thiosulfate, calcium thiosulfate or potassium thiosulfate, preferably sodium thiosulfate.

In another aspect, is a process for degrading hexaflumuron from waste sea water containing hexaflumuron comprising: a) adjusting the pH of the water to a pH of about 10 with NaOH or KOH, b) adding ozone to the water to achieve a TRO level of about 5 to 7 mg/L; c) after ozonation, incubating the water for a period of about 3 to 18 hours, d) neutralizing the water with a reducing agent selected from a sulfite or thiosulfate, and e) discharging the neutralized water into the environment. In another aspect, the sulfite is sodium sulfite, calcium sulfite or potassium sulfite, preferably sodium sulfite and the thiosulfate is sodium thiosulfate, calcium thiosulfate or potassium thiosulfate, preferably sodium thiosulfate. In another aspect, is a process for degrading hexaflumuron from waste sea water containing hexaflumuron comprising: a) adjusting the pH of the water to a pH of about 10 with NaOH or KOH, b) adding ozone to the water to achieve a TRO level of about 5 to 7 mg/L; c) after ozonation, incubating the water for a period of about 3 to 12 hours, d) neutralizing the water with a reducing agent selected from a sulfite or thiosulfate, and e) discharging the neutralized water into the environment. In another aspect, the sulfite is sodium sulfite, calcium sulfite or potassium sulfite, preferably sodium sulfite and the thiosulfate is sodium thiosulfate, calcium thiosulfate or potassium thiosulfate, preferably sodium thiosulfate.

In another aspect, is a process for degrading hexaflumuron from waste sea water containing hexaflumuron comprising: a) adjusting the pH of the water to a pH of about 10 with NaOH or KOH, b) adding ozone to the water to achieve a TRO level of about 5 to 7 mg/L; c) after ozonation, incubating the water for a period of about 3 to 6 hours, d) neutralizing the water with a reducing agent selected from a sulfite or thiosulfate, and e) discharging the neutralized water into the environment. In another aspect, the sulfite is sodium sulfite, calcium sulfite or potassium sulfite, preferably sodium sulfite and the thiosulfate is sodium thiosulfate, calcium thiosulfate or potassium thiosulfate, preferably sodium thiosulfate.

In another aspect, is a process for degrading hexaflumuron from waste sea water containing hexaflumuron comprising: a) adjusting the pH of the water to a pH of about 10 with NaOH or KOH, b) adding ozone to the water to achieve a TRO level of about 5 to 6 mg/L; c) after ozonation, incubating the water for a period of about 3 to 24 hours, d) neutralizing the water with a reducing agent selected from a sulfite or thiosulfate, and e) discharging the neutralized water into the environment. In another aspect, the sulfite is sodium sulfite, calcium sulfite or potassium sulfite, preferably sodium sulfite and the thiosulfate is sodium thiosulfate, calcium thiosulfate or potassium thiosulfate, preferably sodium thiosulfate.

In another aspect, is a process for degrading hexaflumuron from waste sea water containing hexaflumuron comprising: a) adjusting the pH of the water to a pH of about 10 with NaOH or KOH, b) adding ozone to the water to achieve a TRO level of about 5 to 6 mg/L; c) after ozonation, incubating the water for a period of about 3 to 18 hours, d) neutralizing the water with a reducing agent selected from a sulfite or thiosulfate, and e) discharging the neutralized water into the environment. In another aspect, the sulfite is sodium sulfite, calcium sulfite or potassium sulfite, preferably sodium sulfite and the thiosulfate is sodium thiosulfate, calcium thiosulfate or potassium thiosulfate, preferably sodium thiosulfate.

In another aspect, is a process for degrading hexaflumuron from waste sea water containing hexaflumuron comprising: a) adjusting the pH of the water to a pH of about 10 with NaOH or KOH, b) adding ozone to the water to achieve a TRO level of about 5 to 6 mg/L; c) after ozonation, incubating the water for a period of about 3 to 12 hours, d) neutralizing the water with a reducing agent selected from a sulfite or thiosulfate, and e) discharging the neutralized water into the environment. In another aspect, the sulfite is sodium sulfite, calcium sulfite or potassium sulfite, preferably sodium sulfite and the thiosulfate is sodium thiosulfate, calcium thiosulfate or potassium thiosulfate, preferably sodium thiosulfate.

In another aspect, is a process for degrading hexaflumuron from waste sea water containing hexaflumuron comprising: a) adjusting the pH of the water to a pH of about 10 with NaOH or KOH, b) adding ozone to the water to achieve a TRO level of about 5 to 6 mg/L; c) after ozonation, incubating the water for a period of about 3 to 6 hours, d) neutralizing the water with a reducing agent selected from a sulfite or thiosulfate, and e) discharging the neutralized water into the environment. In another aspect, the sulfite is sodium sulfite, calcium sulfite or potassium sulfite, preferably sodium sulfite and the thiosulfate is sodium thiosulfate, calcium thiosulfate or potassium thiosulfate, preferably sodium thiosulfate.

DESCRIPTION

Figures:

Figure 1 . Closed Loop Circulation System

Figure 2. Open Circulation System

Figure 3. Development of TRO and Hexaflumuron Degradation as a Function of Ozone Figure 4. Hexaflumuron Concentration and TRO as a Function of Incubation Time in

Water Collected at TRO 5 mg/L at Different Temperatures Figure 5. Hexaflumuron Concentration and TRO as a Function of Incubation Time in Water Collected at TRO 8 mg/L at Different Temperatures

Figure 6. Development of TRO During Ozonation

Sea lice are parasitic crustaceans/copepods within the order Siphonostomatoida, family Caligidae that feed on the mucus, epidermal tissue, and blood of host marine fish. Sea lice that affect salmon are within the Lepeophtheirus or Caligus species, specifically L salmonis, C. celmensi, C. curtus, C. dussumieri, C. elongates, C. longicaudatus, C. rogercresseyi and C. stromii. Sea lice are prevalent parasites, particularly on salmonids, and, when present in high numbers, can cause welfare issues, serious disease and ultimately, host death. Fish farms usually have high concentrations of fish and a sea louse infestation can have a devastating effect. In fact, many salmon producing countries have legislation limiting the allowed number of sea lice per fish due to welfare and/or salmon stocks.

“Fish” as used herein include fish of all ages in sea water (e.g., marine) and/or brackish water, and more particularly, farmed sea water fish. Non-limiting examples of farmed fish include salmon and trout in the Salmonidae family, sea bream in the Sparidae family and sea bass in the Serranidae family. The preferred fish are salmonid, for example, Salmo salar (Atlantic salmon); S. trutta (brown or sea trout); Oncorhynchus mykiss (rainbow trout); and the Pacific salmon (0. gorbuscha 0. keta; 0. nekra; 0. kisutch, 0. tshawytscha and 0. mason). The more preferred fish is the Atlantic salmon.

Fish can be treated with hexaflumuron by adding hexaflumuron or a hexaflumuron formulation to the sea water containing fish to be treated, for example, by adding hexaflumuron directly to a pen sheltered by a tarpaulin or directly to water in a well boat tank, land-based tank, floating tank, helixir tank or anesthesia cradle. The hexaflumuron can be added to the water from a high concentration stock solution, for example PHARMAQ Alpha Flux® 100 mg/mL, for dilution to about 2 ppm hexaflumuron. To limit the release to the environment of hexaflumuron after bath treatments, or release of other active ingredients administered by bath, it will be highly beneficial to minimize or eliminate the parasiticide from the waste sea water prior to discharging the water into the environment. Sea water, or salt water, is water from an ocean or sea. Sea water can also be prepared by mixing fresh water with a complex of salts, organic and inorganic materials. On average, sea water in the world’s oceans has a salinity of about 3.5% (35 g/L or 35 ppm), from dissolved salts, predominantly sodium chloride. Small amounts of other substances include, for example Mg²⁺, SO4²', Ca²⁺, K⁺, Br, F-, and many other elements. Sea water is not uniformly saline throughout the world. Sea water pH typically ranges from about 7.5 to 8.4 with an average pH of about 8.1 .

Waste sea water, as described herein, refers to sea water that recently housed or transported fish and has been treated with hexaflumuron for treating fish against infestation with sea lice.

A well boat is a vessel with a well(s) or tank(s) for the storage and transport of live fish and are generally used extensively in the aquaculture industry. Each boat generally contains 1 , 2, or 3 wells. Each well can hold about 1000-3000m³ [1 m³ = 1000L] of sea water. These vessels can be used to transport smolt to sea sites, bring smolt and fish to processing sites and/or to sort and delouse live fish. All well boats are equipped with their own oxygen (O2) production equipment for supplying oxygen-rich water to the fish. Ozone (O3) can be generated from pure oxygen in a second step and is commonly used as a part of the disinfection routine of the on-board wells and water transfer lines. Most if not all modern well boats contain ozonation equipment. Ozone equipment can be retrofitted onto older well boats.

When sea water is ozonated, bromide ions (Br) in the water catalytically decompose ozone through several reaction pathways, producing reaction products such as hypobromite (BrO-), hypobromous acid (HOBr), bromate (BrOs-) and bromoform (CHBrs-). These bromo reactants, particularly HOBr, are highly oxidative and are presumed to play a role in hexaflumuron degradation and are longer lived oxidants than ozone. Thus, hexaflumuron degradation in sea water exploits a longer degradation period with lower amounts of ozone, particularly since ozone is rapidly depleted. Concentrations of bromine can be expressed as TRO. Literature indicates that BrO' and HOBr are toxic and have been characterized as such in ozonated sea water.

Lethal concentrations (LCso values) for fish and invertebrates range from TRO values of about 0.015 - 1 .5 mg/L when measured as mg Br2 per liter (Cooper et.al. , 2002). This corresponds to about 0.034 - 3.4 mg/L when TRO is measured as mg of CI2 per liter. TRO is a measure of the oxidative reduction potential in the water and the TRO values described herein for hexaflumuron degradation are expressed by chlorine.

Hypobromite, HOBr and BrOs’ are neutralized when adding a reducing agent, such as sulfite or thiosulfate to the ozonated sea water. Chloro oxidants are also reduced. For purposes of ozonation and degradation of hexaflumuron, a TRO level of about 4 to 9 mg/L is preferred. A more preferred TRO level for degradation of hexaflumuron from ozonation is about 5 to 8 mg/L. An even more preferred TRO level for degradation of hexaflumuron from ozonation is about 5 to 7 mg/L or 5 to 6 mg/L. A preferred TRO level for degradation of hexaflumuron from ozonation is about 5 mg/L. Another preferred TRO level for degradation of hexaflumuron from ozonation is about 5.5 mg/L. Another preferred TRO level for degradation of hexaflumuron from ozonation is about 6 mg/L. Several studies have shown that the development and decay of TRO in sea water during and after ozonation are influenced by organic load, content of nitrogen, salinity, temperature and pH; any and all of which can vary depending on location, tidal activity, current, season (e.g., summer or winter) and fish density.

In addition to well boats, there are also land-based salmon farms using the advanced recirculating aquaculture system (RAS). Within these RAS systems, fish are farmed in a controlled environment based on state-of-the-art recirculation technology that ensures stability of water (e.g., pH, salinity, current flow, temperature, oxygenation and organic matter) with a reduced feed conversion ratio and improved fish survival. In this instance, if and when fish are treated against sea lice with hexaflumuron, subsequent water treatment can be accomplished similarly to that on a well boat. Once fish have been transferred from the treated tank, the waste water can be filtered to remove organic matter, pH adjusted to about 10, infused with ozone to achieve a TRO of about 5-8 mg/L and then incubated for a period of about 3 hours, or more. After the incubation period, a reducing agent, for example sodium sulfite or sodium thiosulfate can be added to the water to neutralize the bromo and/or chloro oxidants. Subsequently, the waste water can be safely discharged into the environment. The land-based and marine based fish farms produce metric tonnes of salmon, bream and bass annually. Hence, there is a need to degrade hexaflumuron from the water after fish treatment prior to discharging the water back into the environment. Ozonation (ozonisation) is a chemical water treatment technique based on the dissolution of ozone into water. Ozone is a triatom ic form of oxygen (O3) gas that is about 10-13x more soluble in water than oxygen (O2). Ozone is a powerful oxidant. In water, ozone has a short half-life (seconds to minutes) that is dependent on pH, temperature, salinity, organic load and availability of O3 scavengers like carbonate. Ozone can be generated through a number of commercially available ozone generators that are reliable and have a low energy cost. Ozone generators work by electrifying air to split oxygen molecules into single atoms. These single atoms then attach to other oxygen molecules to form ozone. Ozone can also be prepared using ultraviolet radiation at a wavelength of about 185 nm, albeit with lower outputs. Depending on oxygen flow rate (liters (or gallons) per minute (L(G)PM)) ozone generators can produce about 45 to 85 g/hr or 35 to 140 g/m³ ozone at a flow rate of between about 4 to 14 LPM. Some ozone generators can produce upwards of about 300 g/m³ (300 mg/L; 300 ppm) ozone. The high ozone concentration produced together with the high gas pressure from these generators results in improved efficiency when dissolving ozone in water. Ozone can be supplied to the water source for dissolution via bubble diffusion, static mixing and injection. Ozone concentration is the ratio of total feed-gas to ozone production. As flow rate of the feed-gas through an ozone generator decreases, ozone concentration increases because the lower flow rate allows more time for ozone generation. Most ozone generators are modular and can be operational in a short period of time. The two most common metrics are ozone output (g/hr) and ozone concentration (g/m³). Ozone conversions for ozone in water include: 1 ppm = 1 mg/L = 1 g/m³; 1 g/hr = 1000 mg/hr; 18.89 g/hr = 1 Ib/day. Other conversion factors include: 1 GPM = 3.78 LPM; and 1 m³ = 264.17 gallons = 998.56 liters. Ozone amounts and concentrations can be calculated using any number of calculations, for example: #1 . (GPM x 3.78 x 60 x ppm) / 1000 = g/hr; #2. (g/hr x 1000) / (GPM x 3.78 x 60) = ppm; and #3. (LPM x 60) x 0.001 ) x g/m³ = g/hr.

Hexaflumuron (N-((3,5-dichloro-4-(1 ,1 ,2,2-tetrafluoroethoxy)phenyl)carbamoyl)- 2,6-difluorobenzamide), a benzoylurea, is a chitin synthesis inhibitor that is used to treat fish infested with sea lice.

A commercial product, Alpha Flux® (PHARMAQ) contains a concentrated (100 mg/mL) solution of hexaflumuron formulated for dilution in water. This concentrated amount of hexaflumuron is added directly to large volumes of water to treat fish against sea lice. A therapeutically effective amount of hexaflumuron to treat fish is about 1 to 5 mg/L (1 -5 ppm), preferably about 2 mg/L (2 ppm). At this concentration (2ppm), fish are treated for a period of up to about 1 to 2 hours. After treatment, the waste sea water can be discharged into the environment. However, due to the physico-chemical characteristics of hexaflumuron, for example, low water solubility, high lipophilicity and molecular structure, hexaflumuron is expected to settle in the sediment with a gradient concentration expanding outward from the fish farm. This sediment may be detrimental to bottom dwelling crustaceans. Therefore, there is a need for reducing and/or eliminating the hexaflumuron from the waste sea water (or fresh water) prior to being discharged into the environment.

Following treatment with hexaflumuron and removal of the fish from the treated sea water, now considered waste sea water, the pH of the waste sea water is adjusted to a pH of about 10 followed by subsequent ozonation to a TRO level of about 4 to 9 mg/L; or about 5 to 8 mg/L; or about 5 to 7 mg/L; or about 5 to 6 mg/L. Depending on the fish density of the tank during treatment, the waste sea water can be filtered through mechanical filters to minimize solid organic matter from the fish. Once the TRO level is achieved, ozonation can cease and the waste sea water is then allowed to incubate for about 3 hours, or more; or about 3 to 120 hours; or about 3 to 48 hours; or about 3 to 24 hours; or about 3 to 18 hours; or about 3 to 12 hours; or about 3 to 6 hours. During this incubation period, residual ozone (if any) and the bromo (and chloro) oxidants continue to degrade the hexaflumuron. Ozonation and incubation leads to sufficient degradation (>90%) of hexaflumuron in the waste sea water. Due to the toxicity of hypobromite and hypobromous acid, these oxidants are then neutralized after the incubation period with a reducing agent (e.g., sulfite, bisulfite or thiosulfate) being added to the water before discharging the waste sea water into the environment. The preferred sulfites are sodium sulfite, potassium sulfite and calcium sulfite. The preferred thiosulfates are sodium thiosulfate, potassium thiosulfate and calcium thiosulfate. The bisulfites, for example sodium bisulfite, potassium bisulfite and calcium bisulfite, can also be used as a reducing agent.

Reducing agents are compounds that lose or donate an electron to an electron recipient in a redox chemical reaction. The agent is typically in one of its lower possible oxidation states and is known as the electron donor. Non-limiting examples of reducing agents include the earth metals, sodium hydride, calcium hydride, lithium aluminum hydride, formic acid, oxalic acid, dithionates, phosphites, sulfites, bisulfites, thiosulfates and the like. Preferred reducing agents include the sulfites, bisulfites and thiosulfates. A preferred sulfite includes sodium sulfite, potassium sulfite, magnesium sulfite and calcium sulfite. A more preferred sulfite is sodium sulfite. A preferred thiosulfate is sodium thiosulfate, potassium thiosulfate, magnesium thiosulfate and calcium thiosulfate. A more preferred thiosulfate is sodium thiosulfate. A commercial product, Ballastguard SBS 40 is a bisulphite based liquid product designed to be used for ballast water treatment systems to neutralize the TRO or total chlorine that can be used as well. In addition to the organic and inorganic reducing agents, bromates can also be reduced by ultraviolet light via photocatalysis alone or in combination with a reducing agent(s).

EXPERIMENTAL

The objectives of the following studies were to assess water sourcing, water quality, temperature, TRO level, ozone concentration and incubation time on hexaflumuron degradation in waste sea water. Methods:

Hexaflumuron was measured using a validated LC-MS/MS method. In short, the method is based on QuEChERS (EN 15662) [quick, easy, cheap, effective, rugged and safe], a type of dispersive solid phase extraction (dSPE) used for sample preparation which is one of the most widely used extraction methods for pesticide analysis. The test kits can be purchased from companies like ThermoFisher® Scientific (Waltham, Massachusetts), Agilant® (SantaClara, California) and BioComma® Limited (Guangdong, China). The kits contain pre-packaged, ready-weighed salts, sorbents and buffers. Hexaflumuron was extracted with acetonitrile before transferring the sample to the LC-MS/MS for analysis. Samples were injected into a LC-system with separation on a C-18 column and programmed mobile phase gradient. Hexaflumuron was ionized with electrospray and analyzed in a mass spectrometer (MS) in MRM mode.

TRO was measured using the colorimetric HACH® Method 8167 test, measured with a HACH DR300 handheld colorimeter following the manufacturer’s instructions. A DPD (N,N-diethyl-p-phenylenediamine) “total chlorine test” (8167) was used to indirectly measure dissolved ozone levels as total residual oxidants (TRO). The results were reported in TRO (mg/L as CI2). TRO can also be measured by total bromide. The two tests are correlated by an atomic mass conversion factor of 2.25. For example, X mg/L Br2 = X / 2.25 = Y mg/L CI2). In short, chlorine/bromine in the water oxidize iodide to iodine. The iodine and free chlorine/bromine reacts with the DPD to form a red solution. The color intensity is proportional to the chlorine/bromine (total residual oxidants) concentration. The value was reported according to a chlorine standard.

Ozone was generated with a Primozone GM3 connected to pressurized oxygen (>99% purity). A Mazzei injector was used to deliver ozone gas into the water column.

A closed circulation pilot scale ozonation system (Figure 1 ) was developed to mimic the ozonation process on a well boat. Ozone production can be varied. The circulating water volume is about 1300-1400 L with a pressure of about 2-3 bar prior to ozonation. Hexaflumuron is added and mixed in IBC Tank 1 . After mixing, water is pumped into tank 2 and through an ozone generator with continual circulation. To mimic and open water tank farm, the open circulation system (Figure 2) was developed. Ozonated water circulates through the open tank using a static mixer and flow pump.

Initially, the closed loop circulation system was used to assess the effect of water temperature on the development of TRO and hexaflumuron degradation in sea water. Approximately 1400 L of sea water was added to the system and hexaflumuron was added to the mixing tank (Tank 1 ) to achieve a hexaflumuron concentration of about 2 ppm. Initial pH of the water was 8.14 which was adjusted to pH 9.94 by addition of 4M NaOH (645 mL) after addition of the hexaflumuron. Water temperature was 11 ,4°C. Ozone generation was set at 34 g/hr giving an input of ozone of 23.5 mg/L/hr. When TRO levels reached the level of between about 5 and 8 mg/L, two or three 1 L water samples were withdrawn from the circulation tank (Tank 2) and were incubated at 4°C (refrigerator), 10°C (water bath, only TRO level (5 mg/L) samples) and 15°C. After incubation periods of approximately 3, 6, 18 and 24 hours, TRO (CL mg/L) was measured according to HACH test 8167 and samples for hexaflumuron were collected by transferring 9.8 mL of ozonated water to 50 mL centrifuge tubes containing 0.2 mL 0.1 M sodium thiosulfate to stop the ozonation/bromine degradant reactions. In the field trial, the amount of ozone added was about 2 mg/L/hr with an ozonation time of about 4.5 hours in total to reach a TRO of about 5 mg/L.

As can be observed in Figure 3, as ozone concentration in the water increases there is a noticeable amount of hexaflumuron degradation, particularly when the level of TRO reaches a level of about 5 to 8 mg/L which in this experiment is observed at an ozone input of about 20-28 mg/L.

TRO 5

When the ozonated water reached a TRO level of about 5 mg/L, three 1 L water samples were collected and incubated at 4, 10 and 15°C. TRO and hexaflumuron concentration was measured at approximately 0, 3, 6, 18 and 24 hours of incubation and is shown below in Table 1 and Figure 4. Percent hexaflumuron degradation amounts are based on an initial hexaflumuron concentration of 1 ,851 pg/L (1.85 ppm). Table 1. Development of TRO (mg/L) and Hexaflumuron Concentration (pg/L) and Degradation (%) at Different Temperatures during Incubation when Ozonating to TRO of about 5 mg/L

As can be seen from Table 1 and Figure 4, both the hexaflumuron concentration and the TRO levels decrease as a function of the incubation period. After 3 hours of incubation, more than 79% of the initial concentration of hexaflumuron degraded at all temperatures. During a 24 hour incubation period, the TRO decreased from 5.1 mg/L to 3.7, 3.2 and 1 .7 mg/L at 4°C, 10°C and 15°C, respectively. During the same time period, the hexaflumuron concentration decreased from 675 pg/L to 109.4 pg/L at 4°C, 17.4 pg/L at 10°C and 4.4 pg/L at 15°C. Hexaflumuron degradation was greater at the warmer water temperatures.

TRO 8

Water was further ozonated until reaching a TRO level of about ~8 mg/L, then two 1 L water samples were collected and incubated at 4°C and 15°C. TRO and hexaflumuron concentration was measured at approximately 0, 3, 6, 18 and 24 hours of incubation; and is shown in Table 2 and Figure 5. Percent hexaflumuron degradation amounts are based on an initial hexaflumuron concentration 1 ,851 pg/L (1.85 ppm) Table 2. Development of TRO (mg/L) and Hexaflumuron (pg/L) Concentration and Degradation (%) at Different Temperatures during Incubation when Ozonating to TRO of about 8 mg/L

As can be seen in Table 2 and Figure 5, both the hexaflumuron concentration and the TRO levels decreased as a function of the incubation period. After 3 hours of incubation, more than 85% of the initial concentration of hexaflumuron degraded at 4°C and 15°C. During the 24 hour incubation period, TRO decreased from 7.7 mg/L to 6.1 and 3.7 mg/L at 4° and 15°C, respectively. During the same period, the hexaflumuron concentration decreased from 481 .4 pg/L to 92.4 pg/L at 4°C and 3.3 pg/L at 15°C.

Overall, measurements of TRO during incubation showed that the rate of TRO depletion was slower at lower temperature. The data also indicate that the rate of TRO depletion is faster when starting incubation at TRO 8 as compared to TRO 5. During the same incubation period, concentration of hexaflumuron in the water samples showed a similar trend, where the rate of hexaflumuron concentration decay over a period of 24 hours was higher at higher temperatures. As with the TRO data, the measured concentration of hexaflumuron decreased with increasing temperature at any given timepoint during the incubation. These data suggest that there is a clear correlation between the temperature, TRO depletion and hexaflumuron degradation. It is noted however, that the correlation between these two variables is not linear which further suggests that other variables in addition to the TRO also play a role in terms of describing the process of hexaflumuron degradation.

Subsequent to the temperature studies described above, a number of pilot scale studies using water from various Norwegian fjord sources, quality, ozone concentration and incubation periods were carried out in either the closed (Figure 1 ) loop circulation or open (Figure 2) circulation systems to optimize the degradation of hexaflumuron from waste sea water adjusted to pH 10. An overview of the pilot study designs is described in Table 3. Table 3. Overview of Pilot Scale Study Designs

Study A

In Study A, 68 mg/L ozone followed by an incubation period of 3 hours resulted in 94.8% hexaflumuron degradation; a >90% hexaflumuron degradation was also obtained for 22 mg/L ozone followed by an incubation period of 4 hours and 11 mg/L ozone followed by an incubation period of 26 hours. Study A showed that a process using lower doses of ozone combined with longer incubation times could achieve high levels of hexaflumuron degradation. During this study, the redox potential of the water was measured in millivolts (mV) rather than TRO; which was shown not to be a suitable measure. Therefore, in subsequent pilot studies, the HACH 8167 colorimetric test was used to measure the level of TRO.

Study B

In Study B, water from a facility rearing salmon in tanks was used. The water was collected at the outlet from a tank holding Atlantic salmon at a density of 13 kg/m³. The salinity of the water was about 3.1 %. Samples of effluent water were un-filtered (Test 1 ) or filtered with either a single 100 micron polypropylene bag filter (Test 2) or a combination of a 100 micron and a 10 micron polypropylene bag filter (Test 3) to assess the impact of extraneous organic matter on TRO and/or hexaflumuron degradation. Data from Pilot Study B is shown below in Table 4. Degradation of hexaflumuron during incubation is shown for three different amounts of added ozone. The higher concentration of ozone equates to a higher TRO, and thus greater hexaflumuron degradation during the ozonation phase before incubation (0 hour). During incubation, the degradation of hexaflumuron continued and reached 80% after 3 hours of incubation and 96% after 7 hours of incubation. In Test 1 the incubation time needed to reach >90% degradation was longer than in the filtered Tests; possibly resulting from the lower level of circulating organic matter in the filtered water. About 95-100% hexaflumuron degradation was reached after about 24 hours incubation in all Tests. The water temperature during ozonation was 9.5°C in Test 1 and 2 and 15°C in Test 3. However, the temperature difference did not appear to influence the TRO level. As can be observed in Table 4, degradation of hexaflumuron after ozonation to TRO ~ 1.5 - 11 mg/L reached 95-100% degradation after 22 -26.5 hours incubation.

Table 4. Study B Hexaflumuron Degradation (%) and Different Incubation Times (hr)

Study C In Study C, further testing of the importance of the water quality for the process was conducted. To obtain a realistic water quality, the open circulation pilot scale experimental unit was brought to a site where fish were treated with hexaflumuron in tanks on land with a fish density of 30 kg/m³. The water temperature was 4.5°C and fish had been kept in the tanks for 2 days with constant water flow prior to the experiment. Total organic content (TOC) in the fish tank water was 13-14 mg/L compared to control sea water with a TOC of 1 .4 mg/L. Some technical problems arose with ozonation, hence the correlation of ozone addition and TRO development was uncertain. Addition of ozone at 30 mg/L was needed to achieve a TRO of about 5 mg/L. The increased amount of ozone needed was a result of a technical issue with the ozone generator that produced a lower than expected output of ozone. In addition, a delay in TRO development was also noted, possibly due to slower rise in ozone concentration, TOC and excess nitrogenous compounds from the fish which is known to cause a lag time in the formation of bromate. When the water was ozonated to a TRO level of 5.75 mg/L, only about 40% of the hexaflumuron was degraded during ozonation; however, after about 18 and 24 hours of incubation, hexaflumuron degraded by about 82% and 94%, respectively.

Study D

In Study D, a water quality with high organic load was studied. Water was collected from a fish transport (Tank 3) with a fish density of 234 kg/m³ water and a TOC of 7.8 mg/L which was compared with control sea water (Tank 1 ). As shown in Figure 6, the TRO level increased with a higher rate in the control sea water compared to the transport water. The observed delay in the increase of TRO levels was similar to that seen in Study C. To reach a TRO level of 5 mg/L, control sea water required addition of about 27 mg/L ozone, while the transport water required > 80 mg/L ozone.

Study E

In Study E, a relevant water sample was obtained with kept fish at a high density of 100 kg/m³ for 2 hours. The fish tank had a partial water change of about 1-2% per hour. Water from the kept fish tank was collected into separate tanks. Tank 1 and Tank 3 contained the waste fish water, Tank 2 contained half waste fish water with remainder control sea water for a prepared 50% density waste sample. Tank 4 contained the control sea water. Prior to ozonation, water samples were pH adjusted to 10. After ozonation (16-23 mg/L) and obtaining a TRO level of about 5 mg/L, samples were incubated. Incubation temperatures (4°C* and 15°C) were varied per tank over the incubation period. TRO level development is shown Table 5.

Table 5. TRO (mg/L) levels at Different Incubation Temperatures over Time

As shown in Table 5, TRO levels showed a similar depletion rate in tank 1 , 2 and 4, whereas the depletion rate in tank 3 was lower. The sample from tank 3 was incubated directly at about 4°C, whereas the samples from the other tanks were kept at ambient conditions during the first part of the incubation. The temperature affected both the rates of TRO depletion and degradation of h exafl umuron. After 5 hours, hexaflumuron degradation in tanks 1 , 2 and 4 was >97%. In tank 3 the hexaflumuron degradation was slower with values of about 87%, 94% and 99% after 7, 24 and 120 hours, respectively.

Overall, hexaflumuron can be almost completely degraded from treated waste sea water by adjusting the pH of the waste sea water to about 10, ozonating the water to a TRO level of about 5 mg/L followed by incubation of the water until sufficiently degraded (>90% for a period of about 3 hours). The amount of ozone needed to reach the desired TRO level is influenced by the water quality. Since well boats have the capacity for oxygen production, an ozone generator can be fitted on the boats to achieve ozonation of 5 mg/L/hr to achieve a TRO level of about 5 mg/L. Degradation of hexaflumuron starts during ozonation of the waste sea water and continues thereafter as a result of the bromate oxidants. Hexaflumuron degradation rate is temperature dependent, with lower rates correlating with lower temperatures. Increased ozonation may be required to achieve higher TRO levels and/or longer incubation times may be required to sufficiently degrade hexaflumuron at lower temperatures. Prior to discharging the water into the environment, a reducing agent, for example sodium sulfite or sodium thiosulfate can be added to the waste sea water to neutralize the bromate degradants. Toxicology

To ensure that the effluent from the degradation process did not have any unacceptable environmental effects, whole effluent toxicity (WET) tests for acute toxicity against the marine crustacean Tisbe battagliai and the marine algae Skeletonema costatum were conducted. Effluent samples were saved and frozen from some of the studies described herein. Upon thawing, salinity was adjusted to about 2.8-3.2% and sodium sulfite was added prior to freezing. No toxic effects were observed for T. battagliai during 48 hours exposure to the effluent. Two tests with S. costatum followed growth of the algae for 72 hours. The Lowest Observed Effect Concentration (LOEC) was registered within the range of 0% (un-diluted) to 50% diluted effluent. The No Observed Effect Concentration (NOEC) was within the range of 50-75% diluted effluent. During commercial use, effluent will be discharged from a moving well boat thereby further diluting the effluent which will be low risk and well below the predicted NOEC for S. costatum.

Claims

CLAIMS We claim:

1 . A process for degrading hexaflumuron from waste sea water containing hexaflumuron comprising: a. adjusting the pH of the water to a pH of about 10; b. adding ozone to the water to achieve a TRO level of about 4 to 9 mg/L; c. after ozonation, incubating the water for about 3 hours, or more; d. after incubation, neutralizing the water with a reducing agent; and e. discharging the neutralized water into the environment.

2. The process of Claim 1 wherein the pH is adjusted with sodium hydroxide or potassium hydroxide.

3. The process of Claim 1 , wherein the amount of ozone added to the water is about 10 to 30 mg/L.

4. The process of Claim 1 , wherein the TRO level is about 5 to 8 mg/L and the sea water is incubated after ozonation for a period of about 3 to about 48 hours.

5. The process of Claim 4, wherein the TRO level is about 5 to 7 mg/L, the reducing agent is a sulfite, bisulfite or thiosulfate.

6. The process of Claim 5, wherein the TRO level is about 5 to 6 mg/L and the incubation period after ozonation is about 3 to about 12 hours.

7. A process for degrading hexaflumuron from waste sea water containing hexaflumuron comprising: a. adjusting the pH of the water to a pH of about 10 with a base; b. adding ozone to the water to achieve a TRO level of about 5 to 7 mg/L; c. after ozonation, incubating the water for a period of about 3 to about 48 hours; d. after incubation, neutralizing the water with a reducing agent selected from a sulfite, bisulfite or thiosulfate; and e. discharging the neutralized water into the environment.

8. The process of Claim 7, wherein the amount of ozone added to the water is about 10 to 30 mg/L.

9. The process of Claim 8, wherein the base is sodium hydroxide or potassium hydroxide; and the TRO level is about 5 to 6 mg/L.

10. The process of Claim 9, wherein the incubation period is about 3 to about 24 hours; and the reducing agent is selected from the group consisting of sodium sulfite, potassium sulfite, calcium sulfite, sodium thiosulfate, potassium thiosulfate and calcium thiosulfate.

11 . The process of Claim 10, wherein the incubation period is about 3 to about 12 hours.

12. The process of Claim 11 , wherein the reducing agent is sodium sulfite or sodium thiosulfate.

13. A process for degrading hexaflumuron from waste sea water containing hexaflumuron comprising: a. adjusting the pH of the water to a pH of about 10 with a base; b. adding ozone to the water to achieve a TRO level of about 5 to 6 mg/L; c. after ozonation, incubating the water for a period of about 3 to about 24 hours; d. after incubation, neutralizing the water with a reducing agent; and e. discharging the neutralized water into the environment.

14. The process of Claim 13, wherein the base is sodium hydroxide or potassium hydroxide; and the amount of ozone added to the water is about 10 to 30 mg/L; and after ozonation, inclubating the water for about 3 to about 12 hours; and after incubation neutralizing the water with a reducing agent selected from the group consisting of sodium sulfite, potassium sulfite, calcium sulfite, sodium thiosulfate, potassium thiosulfate and calcium thiosulfate.

15. The process of Claim 14, wherein the reducing agent is sodium sulfite or sodium thiosulfate.