DK202470076A1

DK202470076A1 - Degradation of isoxazolines in ozonated sea water

Info

Publication number: DK202470076A1
Application number: DKPA202470076A
Authority: DK
Inventors: Croton Claudia; Johnsen ØSTNÆS Simen; Rode Marit
Original assignee: Zoetis Services Llc
Priority date: 2023-03-13
Filing date: 2024-03-07
Publication date: 2024-10-10
Also published as: WO2024192071A1; DK182016B1

Abstract

The invention describes a process for degrading an isoxazoline from waste sea water treated with an isoxazoline that was used to treat fish against sea lice infestation by optionally adjusting the pH of the water to a pH range of about 9-10 with a base; ozonating the water to achieve a total residual oxidant (TRO) level of about 2 to 9 mg/L; preferably, 3 to 8 mg/L; after ozonation, incubating the water for a period of 0 to about 24 hours; 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

DK 2024 70076 A1 1

DEGRADATION OF ISOXAZOLINES IN OZONATED SEA WATER

FIELD OF THE INVENTION

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

BACKGROUND OF THE INVENTION

Isoxazolines are ectoparasitic compounds that are GABA-gated chloride channel antagonists. The isoxazolines are generally used for land-based ectoparasites including insects (e.g., fleas, mites, lice and mosquitos) and arachnids (e.g., ticks). Some isoxazolines are also being developed for treating fish against sea lice. As such, there exists a need for treating waste sea water that contains an isoxazoline to minimize and/or eliminate the isoxazoline prior to discharging the water back into the environment.

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 or other agent. 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., diflubenzuron, hexaflumuron) or hydrogen peroxide are common antiparasitic immersion treatments.

New treatments may include the use of an isoxazoline.

DK 2024 70076 A1 2

Frozen and thawing 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 freshwater 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 an isoxazoline from treated waste sea water following treatment of live fish with an isoxazoline 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 an isoxazoline from the waste sea water after fish treatment before discharging the water into the environment.

DK 2024 70076 A1 3

SUMMARY OF THE INVENTION

In one aspect of the invention, is a process for degrading an isoxazoline from waste sea water containing an isoxazoline comprising: a) otionally adjusting the water to a pH range of 9-10 with a base; b) adding ozone to the water to achieve a Total

Residual Oxidants (TRO) level of about 2 to 9 mg/L or 3 to 8 mg/L; c) after ozonation, optionally incubating the water for about 1 or more hours; d) neutralizing the water with a reducing agent; and e) discharging the neutralized water into the environment. In another aspect of the invention, is a process for degrading an isoxazoline from waste sea water containing an isoxazoline comprising: a) adjusting the pH of the water to a pH ranging from about 9 to 10; b) adding ozone to the water to achieve a Total Residual

Oxidants (TRO) level of about 2 to 9 mg/L or 3 to 8 mg/L; c) after ozonation, optionally incubating the water for about 1 or more hours; d) neutralizing the water with a reducing agent; and e) discharging the neutralized water into the environment. In another aspect, the waste sea water is adjusted to a pH of about 9 with a base. 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, 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, 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 ozone 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 ozone of about 15 to 28 mg/L ozone. In another aspect, the ozone is added to the waste sea water to achieve an amount of ozone 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 ozone 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

DK 2024 70076 A1 4 vary.

In view of this variable, ozone is added to the waste sea water to achieve a TRO level of about 2 to 9 mg/L.

In another aspect, the ozone is added to the waste sea water to achieve a TRO level of about 3 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 3 mg/L.

In another aspect, the ozone is added to the waste sea water to achieve a TRO level of about 4 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 6 mg/L.

In another aspect, the ozone is added to the waste sea water to achieve a TRO level of about 7 mg/L.

In another aspect, the ozone is added to the waste sea water to achieve a TRO level of about 8 mg/L.

In another aspect, the waste sea water is incubated for a period of about O to 24 hours.

In another aspect, the waste sea water is incubated for a period of about 0 to about 18 hours.

In another aspect, the waste sea water is incubated for a period of about 0 to about 12 hours.

In another aspect, the waste sea water is incubated for a period of about 0 to about 6 hours.

In another aspect, the waste sea water is incubated for a period of about 0 to about 4 hours.

In another aspect, the waste sea water is incubated for a period of about O to about 3 hours.

In another aspect, the waste sea water is incubated for a period of about 0 to about 2 hours.

In another aspect, the waste sea water is incubated for a period of about O to about 1 hour.

In another aspect, the waste sea water is incubated for a period of about 0.1 to about 1 hour.

In another aspect, the waste sea water is not incubated after ozonation to a specific TRO level.

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.

DK 2024 70076 A1

In another aspect, is a process for degrading an isoxazoline from waste sea water containing an isoxazoline comprising: a) adding ozone to the water to achieve a

TRO level of about 3 to 8 mg/L; b) after ozonation, incubating the water for a period of about 0 to 24 hours, c) neutralizing the water with a reducing agent selected from a 5 sulfite or thiosulfate, and d) 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 an isoxazoline from waste sea water containing an isoxazoline comprising: a) adjusting the pH of the water to a pH range of about 9-10 with a base; b) adding ozone to the water to achieve a TRO level of about 3 to 8 mg/L; c) after ozonation, incubating the water for a period of about 0 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 base is sodium hydroxide or potassium hydroxide. 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 an isoxazoline from waste sea water containing an isoxazoline comprising: a) adjusting the pH of the water to a pH of about 9 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 0 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 an isoxazoline from waste sea water containing an isoxazoline 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 0 to 24

DK 2024 70076 A1 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.

TRO level of about 3 to 8 mg/L; c) after ozonation, incubating the water for a period of about 0 to 12 hours, c) neutralizing the water with a reducing agent selected from a sulfite or thiosulfate, and d) discharging the neutralized water into the environment. In another aspect, the sulfite is sodibum 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 an isoxazoline from waste sea water containing an isoxazoline comprising: a) adjusting the pH of the water to a pH range of about 9-10 with NaOH or KOH, b) adding ozone to the water to achieve a TRO level of about 3 to 8 mg/L; c) after ozonation, incubating the water for a period of about 0 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.

TRO level of about 3 to 8 mg/L; c) after ozonation, incubating the water for a period of about 0 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.

DK 2024 70076 A1 7

In another aspect, is a process for degrading an isoxazoline from waste sea water containing an isoxazoline comprising: a) adjusting the pH of the water to a pH range of about 9-10 with NaOH or KOH, b) adding ozone to the water to achieve a TRO level of about 3 to 8 mg/L; c) after ozonation, incubating the water for a period of about 0to 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.

TRO level of about 3 to 8 mg/L; b) after ozonation, incubating the water for a period of about 0 to 3 hours, c) neutralizing the water with a reducing agent selected from a sulfite or thiosulfate, and d) 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 an isoxazoline from waste sea water containing an isoxazoline comprising: a) adjusting the pH of the water to a pH range of about 9-10 with NaOH or KOH, b) adding ozone to the water to achieve a TRO level of about 3 to 8 mg/L; c) after ozonation, incubating the water for a period of about 0 to 3 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.

TRO level of about 3 to 8 mg/L; b) after ozonation, incubating the water for a period of about to 1 hour, c) neutralizing the water with a reducing agent selected from a sulfite or thiosulfate, and d) discharging the neutralized water into the environment. In another

DK 2024 70076 A1 8 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 an isoxazoline from waste sea water containing an isoxazoline comprising: a) adjusting the pH of the water to a pH range of about 9-10 with NaOH or KOH, b) adding ozone to the water to achieve a TRO level of about 3 to 8 mg/L; c) after ozonation, incubating the water for a period of about 0 to 1 hour, 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 an isoxazoline from waste sea water containing an isoxazoline comprising: a) adjusting the pH of the water to a pH of about 9 with NaOH or KOH, b) adding ozone to the water to achieve a TRO level of about 3 to 8 mg/L; c) after ozonation, incubating the water for a period of about O to 1 hour, 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 an isoxazoline from waste sea water containing an isoxazoline 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 3 to 8 mg/L; c) after ozonation, incubating the water for a period of about O to 1 hour, 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.

DK 2024 70076 A1 9

TRO level of about 3 to 8 mg/L; b) after ozonation, neutralizing the water with a reducing agent selected from a sulfite or thiosulfate, and c) discharging the neutralized waterinto 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 an isoxazoline from waste sea water containing an isoxazoline comprising: a) adjusting the pH of the water to a pH range of about 9-10 with NaOH or KOH, b) adding ozone to the water to achieve a TRO level of about 3 to 8 mg/L; c) after ozonation, neutralizing the water with a reducing agent selected from a sulfite or thiosulfate, and d) 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 an isoxazoline from waste sea water containing an isoxazoline comprising: a) adjusting the pH of the water to a pH of about 9 with NaOH or KOH, b) adding ozone to the water to achieve a TRO level of about 3 to 8 mg/L; c) after ozonation, neutralizing the water with a reducing agent selected from a sulfite or thiosulfate, and d) 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 an isoxazoline from waste sea — water containing an isoxazoline 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 3 to 8 mg/L; c) after ozonation, neutralizing the water with a reducing agent selected from a sulfite or thiosulfate, and d) 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.

DK 2024 70076 A1 10

DESCRIPTION

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. dussumiern, 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 Sa/monidae 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 (O. gorbuscha; O. keta; O. nekra; O. kisutch, O. tshawytscha and O. mason). The more preferred fish is the Atlantic salmon.

Fish can be treated with an isoxazoline by adding an isoxazoline to the sea water containing fish to be treated, for example, by adding an isoxazoline 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 isoxazoline can be added to the water from a high concentration stock solution for dilution to about 0.5 to about 250ppb, depending on the isoxazoline. Due to the low solubility of isoxazolines, the compound will generally need to be formulated with a solvent, cosolvent, surfactant, solubility enhancer and/or emulsifier to prepare a concentrated solution for dilution. To limit the release to the environment of an isoxazoline 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.

DK 2024 70076 A1 11

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.

The following three isooxazoline compounds were tested in the ozonation process. The isoxazolines were prepared in accordance with the schemes and methods described in patent publications WO2012/120389 (Formula 1 and 2) and

WO2012/017359 {Formula 3). 0 00 0080 F ry ; ig M

CF; ® © cr (J PP . @® or (1) " (0) oN @ . (J ø

F

Cl F Cl

The Formula (1) compound is ($)-1-(5'-5-(3,5-dichiorophenyl)-5-ftriftuoromethyl)- 4 5-dihydroisoxazol-3-y1)-3'H-spirofazetidine-3. 1'-isobenzofuran]-1-y!)-2- (methylsuffonyhjethan-1-one; the Formula (2) compound is ($)-1-(3'-15-(3-chloro-5- fluorophenyt)-5-(trifluoromethyl)-4 5-dihydroisoxazol-3-yl)-3'H-spirof[azetidine-3, 1- isobenzofuran]-1-yl)-2-(methylsulfonytjethan-1-one; and the Formula (3) compound is {S)-N-cyclopropyi-3-{4-(5-(3 5-dichioro-4-fluoropheny?)-S-(triftuoromethyl)-4 5- dihydroisoxazol-3-ylipheny!)-3-fluoroazetidine-1-carboxamide. As noted, these compounds are all stereoisomers. Racemic mixtures {ie the (S) and (R)) are also contemplated for degradation by ozone according fo the instant invention. Further, isoxazolines, and stereoisomers thereof, described in the following PCT patent z0 application publications (WO) are also contemplated under the general term “‘isoxazoline(s)” and include: WO2012/120399, W2012/017359, WO2007/079162 {e.q., afoxolaner (4-(5-(3-chioro-5-(Irifluoromethyhpheny!)-&-(frifluoromethy!)-4 ,5- dihydroisoxazol-3-yl)-N-(2-0x0-2-((2 2, 2-trifluorcethyhamino)jethyl)- 1 -naphthamide), and the S-enantiomer of afoxolaner (esafoxolaner));, WO2005/085218 {e.g., fluralaner (4-(5- — (3,5-dichlorophenyl)-S-(triffuoromethyl)-4 5-dihydroisoxazol-3-yt)-2-methyl-N-(2-0x0-2-

DK 2024 70076 A1 12 ((2,2,2-trifluoroethyljamino)ethyl)-benzamide)), WO2015/086551, WO2010/070068 (e.g., lotilaner ((S)-3-methyl-N-(2-ox0-2-((2,2,2-trifluoroethyl)amino)ethyl)-5-(5-(3,4,5- trichlorophenyl)-5-(trifluoromethyl)-4,5-dihydroisoxazol-3-yl)thiophene-2-carboxamide)),

WO2022/263573 and WO2022/271961 (e.g., mivorilaner ((S)-3-(5-(3,5-dichloro-4- fluorophenyl)-5-(trifluoromethyl)-4,5-dinydroisoxazol-3-yl)-N-(2-((2,2- difluoroethyljamino)-2-oxoethyl)-5,6-dihydro-4H-cyclopenta[c]thiophene-1- carboxamide)); WO2017/147352 (e.g., umifoxolaner (4-(5-(3-chloro-4-fluoro-5- (trifluoromethyl)phenyl)-5-(trifluoromethyl)-4,5-dihydroisoxazol-3-yl)-N-(2-ox0-2-((2,2,2- trifluoroethyl)amino)ethyl)-1-naphthamide)).

The isoxazoline ozonation process is also contemplated to be used to degrade other similar per-fluoro-structural compounds, for example, tigolaner (1-(2-(2-chloro-5- (2'-methyl-5'-(perfluoroethyl)-4'-(trifluoromethyl)-2'H-1,3'-bipyrazol]-4-yl)phenyl)-2- oxoethyl)cyclopropane-1-carbonitrile), described in WO2021/028479.

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 Mg?*, SO4%, Ca?", K*, Br, F7, 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 an isoxazoline 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 1000-3000m? of sea water. These vessels can be used to transport smolt to sea sites, to bring them to processing sites and/or to sort and delouse live fish. All well boats are equipped with their own oxygen (Oz) production equipment for supplying oxygen-rich water to the fish.

Ozone (Os) 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

DK 2024 70076 A1 13 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 (BrO3) and bromoform (CHBr3). These bromo reactants, particularly HOBr, are highly oxidative and are presumed to play a role in an isoxazoline degradation and are longer lived oxidants than ozone. Thus, an isoxazoline 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 Br» per liter (Cooper et.al., 2002). This corresponds to about 0.034 — 3.4 mg/L when TRO is measured as mg of Cl, per liter.

TRO is a measure of the oxidative reduction potential in the water and the TRO values described herein for an isoxazoline degradation are expressed by chlorine. In short, chlorine/bromine in the water oxidize iodide to iodine. The iodine and free chlorine/bromine reacts with DPD (N,N-diethyl-p-phenylenediamine) to form a red solution. The color intensity is proportional to the chlorine/bromine (= total residual oxidants) concentration. The value is reported according to a chlorine standard.

Hypobromite, HOBr and BrOz 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 an isoxazoline, a TRO level of about 2 to 9 mg/L is preferred. A more preferred TRO level for degradation of an isoxazoline from ozonation is about 3 to 9 mg/L; or 3 to 8mg/L; or 3 mg/L; or 4 mg/L; or 5 mg/L, or 6 mg/L; or 7 mg/L; or 8 mg/L. Also contemplated are fractional TRO levels, for example, 2.8 mg/L, 3.3 mg/L, 4.8 mg/L, 5.3 mg/L, 6.2 mg/L, 7.4 mg/L, 8.3mg/L, and the like.

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.

DK 2024 70076 A1 14

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 for an ectoparasitic infection with an isoxazoline, 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 2 to 9 mg/L and then incubated for a period of about 0 to 24 hours. After the incubation period, if needed, 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, the need to degrade an isoxazoline from the water after fish treatment prior to discharging the water back into the environment.

Ozonation is a chemical water treatment technique based on the dissolution of ozone into water. Ozone is a triatomic form of oxygen (Os) gas that is about 10-13x more soluble in water than oxygen (Oz). 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 Os 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/m? ozone at a flow rate of between about 4 to 14 LPM. Some ozone generators can produce upwards of about 300 g/m? (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

DK 2024 70076 A1 15 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/m). Ozone conversions for ozone in water include: 1 ppm = 1 mg/L = 1 g/m?; 1 g/hr = 1000 mg/hr; 18.89 g/hr = 1 Ib/day. Other conversion factors include: 1 GPM = 3.78 LPM; and 1 m? = 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/m? = g/hr.

Isoxazolines are GABA-gated chloride channel antagonists that can be used to treat fish infested with sea lice. Concentrated solutions of an isoxazoline, for example a

Formula (1) compound, Formula (2) compound or Formula (3) compound, or any other isoxazoline, can be prepared and used for dilution into small or large volumes of sea water to treat fish against sea lice. A therapeutically effective amount of an isoxazoline to delouse fish is about 0.5 to 250 ug/L (0.5-250 ppb); or 0.5 to 200 ppb, or 1 to 200 ppb. Atthese concentrations, fish are treated for a period of up to about 2 hours. After treatment, the fish are removed from the treated tank and returned to their live pens or housed in separate non-treated tanks and the waste sea water was routinely discharged into the environment. However, due to the low water solubility of an isoxazoline, the isoxazoline is expected to settle in sediment with a gradient concentration from the fish farm. This sediment may be detrimental to bottom dwelling crustaceans. Therefore, there is a need for reducing and/or eliminating the isoxazoline from the waste sea water (or fresh water) prior to being discharged into the environment.

Depending on the isoxazoline used to treat fish against sea lice, the waste sea water can be ozonated without adjusting the pH of the water to a TRO of about 3-8 mg/L. In other instances, the waste sea water is pH adjusted with a base prior to ozonation to a TRO level of about 3-8mg/L. If the waste sea water is pH adjusted with a base, it can be adjusted to a range of about 9-10, and preferably to a pH of about 9 or a

DK 2024 70076 A1 16 pH of about 10. A number of bases can be used to increase the alkalinity of the waste sea water. Non-limiting examples of a base include: calcium carbonate, calcium hydroxide, potassium acetate, potassium bicarbonate, potassium hydroxide, sodium acetate, sodium bicarbonate, sodium hydroxide, sodium bicarbonate, trisodium phosphate, and the like. The preferred base is selected from sodium hydroxide and potassium hydroxide.

Following treatment with an isoxazoline and removal of the fish from the treated sea water, now considered waste sea water, the pH of the waste sea water can be adjusted to a pH range of about 9 to 10 followed by subsequent ozonation to a TRO level of about 2 to 9 mg/L; or about 3 to 8 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 desired pH is obtained, the waste sea water is ozonated and the waste sea water can incubate after ozonation for a period of 0-24 hours, or about 0 to 18 hours; or about O to 12 hours; or about O to 6 hours; or about 0 to 3 hours; or about 0 to 1 hour; or not incubated at all after ozonation to a TRO level of about 3-8 mg/L. During ozonation and/or the incubation period (0-24 hours), residual ozone and the bromo (and chloro) oxidants continue to degrade the isoxazoline. Ozonation and incubation (0-24 hours) leads to sufficient degradation (>99%) of an isoxazoline in the waste sea water. Due to the toxicity of hypobromite and hypobromous acid, these oxidants are then neutralized after ozonation and/or the incubation period (0-24 hours) 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

DK 2024 70076 A1 17 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 isoxazoline degradation in waste sea water.

Methods:

An isoxazoline was measured using a validated LC-MS/MS method. In short, a given volume of isopropanol and the internal standard were added to the seawater sample containing the respective isoxazoline. The sample was mixed thoroughly before being transferred to the LC-MS/MS for analysis. Samples were injected into a LC- system with separation on a Biphenyl 2.1x50 mm column (2.7 um particles) and programmed mobile phase gradient (mobile phase A: water with 0.1% formic acid; mobile phase B: methanol with 0.1% formic acid). An isoxazoline 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 Cb). 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

Bro = X / 2.25 = Y mg/L Cl).

DK 2024 70076 A1 18

In an initial study, 10L samples of seawater from fish transport (118kg/m?; 30.7% salinity) at room temperature (21.3°C) were spiked with an isoxazoline of Formula (1), (2) or (3) and incubated with stirring for 1-hour to simulate fish treatment. Concentration for Formula (1) was 20 pg/L (20ppb); concentration of Formula (2) was 100ug/L (100ppb); and Formula (3) was 10ug/L (10ppb). 10L (unadjusted pH (=8)) and 10L (pH adjusted to 10 with 4M NaOH) were ozonated (=4g/hr) and samples of approximately 1

L were withdrawn at TRO levels of about 3, 5 and 8 mg/L. Samples from these containers were withdrawn after 0, 1, 3, 5 and 24 hours. Results of isoxazoline (Formula (1), Formula (2) and Formula (3) degradation at pH 8 and 10 and at different

TRO levels of about 3-8mg/L is described below.

The Formula (1) compound degraded at pH 10 by 98.2% (TRO 3.2mg/L) to >99% at TRO levels of 5.5mg/L and 7.8mg/L at O hours after ozonation. By 1 hour, degradation was = 99% at all TRO levels (2.5-7mg/L). Degradation at pH 8 was lower at 0 hours with degradation amounts of about 79%, 90% and 90% at TRO levels of 3.0 mg/L, 6.2 mg/L and 8.6 mg/L, respectively. At 24 hours (pH 8) after ozonation, degradation was about 87%, 94% and 96% for the lowest to highest TRO levels, respectively.

The Formula (2) compound degraded at pH 10 by about 97.6-99.9% at 0 hours after ozonation at TRO levels of about 3mg/L, 4.9mg/L and 8mg/L. At one hour after ozonation, degradation was 98.2 % (TRO 2mg/L) to >99% at TRO levels of 4.3mg/L and 6.7mg/L. As TRO levels declined overtime at the lower end, degradation reached 99% at 3 hours at TRO of 1.8mg/L. Degradation at pH 8 was lower with % degradation amounts of about 61%, 77% and 93% for the TRO of 3.3 mg/L, 5.7 mg/L and 8.2 mg/l, respectively. At 24 hours (pH 8) after ozonation, degradation was about 82%, 91.3% and 95.2% for the lowest to highest TRO levels, respectively. Similar degradation numbers were observed when conducted in 14°C water with a fish density of 88kg/m? and a pH of 10 prior to incubation. AtO hours after ozonation, Formula (2) degradation at TRO of 2.8mg/mL and 5.1mg/mL was greater than 98%.

The Formula (3) compound degraded at pH 10 by 98.6% at 0 hours after ozonation at a TRO of about 3 mg/L and >99% at TRO levels of 5. 1mg/L and 8. 1mg/L. at the same TRO. After 1 hour, degradation was 299% at all TRO levels. At pH 8,

DK 2024 70076 A1 19 degradation at 0 hours was lower with degradation of about 80% and 96% at a TRO level of 3.2mg/L and 5.5mg/L, respectively, with degradation >99% at TRO 8.2mg/L.

After 1 hour at pH 8, degradation was = 99% at all TRO levels.

Overall, an isoxazoline can be degraded by = 98% from treated waste sea water by adjusting the pH of the waste sea water to about 10, ozonating the water to a TRO level ranging from about of about 3 to 8 mg/L with a 0-hour incubation period after ozonation. Depending on the isoxazoline, TRO level, water quality and lower pH value (=8), longer incubation periods may be needed. 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 the needed TRO levels to degrade an isoxazoline from the waste sea water with an incubation period ranging from 0-24 hours after ozonation.

Degradation of an isoxazoline starts during ozonation of the waste sea water and continues thereafter as a result of the bromate and other oxidants. An isoxazoline degradation rate may be 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 an isoxazoline 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.

After ozonation to a TRO level ranging from about 3 to about 8mg/L, TRO levels slowly erode over time, thereby necessitating the addition of a reducing agent to the waste sea water after the isoxazoline is degraded so that the waste sea water can be discharged into the environment reasonably soon after isoxazoline treatment. The average (n=2%; n=3) TRO level degradation over time for the three isoxazolines tested is shown in Table 1.

DK 2024 70076 A1

Table 1. TRO Degradation After Ozonation ow en 0 1 2 | 58 | 8%

Claims

DK 2024 70076 A1 21 CLAIMS

1. A process for degrading an isoxazoline from waste sea water comprising:

a. optionally, adjusting the pH of the water with a base to a pH of about 9 to about 10;

b. adding ozone to the water to achieve a TRO level of about 2 mg/L to about 9 mg;

€. after ozonation, incubating the water for a period of from 0 to about 24 hours;

d. neutralizing the water with a reducing agent; and e. discharging the neutfralized water into the environment.

2. The process of claim 1, wherein the isoxazoline is selected from the group consisting of a Formula (1), Formula (2) or the Formula (3) compound. O & og Od. | F . NÅ Å + 3 I N N æ CF ® 9 cry ® o Fs 3 . (0) or (1) © (0 SÅ 00 5 9 9 ” Cl F Ci

3. The process of claim 2, wherein the isoxazoline is the Formula (2) compound.

4, The process of claim 1, further comprising adjusting the pH of the water with a base selected from sodium hydroxide or potassium hydroxide.

5. The process of claim 1 or 4, wherein the amount of ozone added to the water is about 10 mg/L to about 30 mg/L and wherein the TRO level is about 3 mg/L to about 8 mg/L and wherein the sea water is incubated after ozonation for a period of 0 to about 12 hours.

DK 2024 70076 A1 22

6. A process for degrading an isoxazoline from waste sea water comprising:

a. optionally, adjusting the pH of the water with a base to a pH range of about 9 to about 10;

b. adding ozone to the water to achieve a TRO level of about 3 mg/L to about 8 mg/L,

C. after ozonation, incubating the water for a period of about 0 to about 12 hours;

d. neutralizing the water with a reducing agent selected from a sulfite, bisulfite or thiosulfate; and e. discharging the neutralized water into the environment.

7. The process of Claim 8, further comprising adjusting the pH with a base and wherein the base is sodium hydroxide or potassium hydroxide, and wherein the amount of ozone added to the water is about 10 mg/L to about 30 mg/L, and the reducing agent is selected from the group consisting of sodium sulfite, potassium sulfite, calcium sulfite, 145 sodium thiosulfate, potassium thiosulfate and calcium thiosulfate.

8. The process of claim 7, wherein the isoxazoline is selected from the group consisting of a Formula (1), Formula (2) or the Formula (3) compound O Sø o 0d, | ef MA J TT CF, (0 9 CF, p 9 CFz X Tet, © (SÅ a KJ : " Cl F Ci

9. The process of claim 7, wherein the water is incubated for a period of 0 to about 6 hours after ozonation.

10. The process of claim 7, wherein the water is incubated for a period of 0 to about 3 hours after ozonation.

DK 2024 70076 A1 23

11. The process of claim 7, wherein the water is incubated for 0 to about 1 hour after ozonation. s

12. Å process for degrading an isoxazoline from waste sea water wherein the isoxazoline is selected from the group consisting of a Formula (1), Formula (2) or Formula (3) compound 0 oo 00 F ry ; > CF, (7 Oo CF ® O CFsa f 4 © D oN cl 0) oN © © (J “a F Cl F Ci comprising:

a. optionally, adjusting the pH of the water fo a pH range of about 9-10 with a base;

b. adding ozone to the water to achieve a TRO level of about 3 mg/L to about 8 mot;

€. after ozonation, incubating the water for a period of 0 to about 6 hours;

d. neutralizing the water with a reducing agent; and e. discharging the neutralized water into the environment.

13. The process of claim 12, further comprising adjusting the pH with a base and wherein the amount of ozone added to the water is about 10 mg/L to about 30 mg/L and zo the reducing agent is selected from the group consisting of sodium sulfite, potassium sulfite, calcium sulfite, sodium thiosulfate, potassium thiosulfate and calcium thiosulfate.

14. The process of claim 13, wherein the base is sodium hydroxide or potassium hydroxide.

DK 2024 70076 A1 24

15. A process for degrading an isoxazoline from waste sea water wherein the isoxazoline is selected from the group consisting of a Formula (1), Formula (2) or Formula (3) compound 0 Nr 6 og. Fe nA N r CF3 | ® ° cr, (0 9 Cs IT Y Cl ® oN n Ci ® ON @ Ci 0 Fx F Cl F Ci comprising:

a. adjusting the pH of the water to a pH range of about 8-10 with a base selected from sodium hydroxide or potassium hydroxide;

b. adding ozone to the water to achieve a TRO level of about 3 mg/L to about 8 mg/L;

Cc. after ozonation, incubating the water for a period of 0 to about 12 hours;

d. 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; and a, discharging the neutralized water into the environment.

g TY DANISH PATENT AND 335 mee TRADEMARK OFFKE Application No. Search report - patent PA 2024 70076

1. [] Certain claims were found unsearchable (See Box No. I).

2. [1] Lack of unity of invention was found prior to search (See Box No. II).

A. Classification CO2F 1/78 (2023.01)i, CO2F 1/72 (2023.01)i, CO2F 1/66 (2023.01)a, CO2F 101/34 (2006.01)a, CO2F 101/36 (2006.01)a,CO2F 103/20 (2006.01)a. According to International Patent Classification (IPC) CO2F 1/78 (2013.01), CO2F 1/72 (2013.01), CO2F 1/66 (2013.01) According to Cooperative Patent Classification (CPC)

B. Fields searched PCT-minimum documentation searched (classification system followed by classification symbols) IPC & CPC:CO2F. Documentation searched other than PCT-minimum documentation DK, NO, SE, FI: IPC-classes CO2F 1/70, CO2F 1/78. Electronic database consulted during the search (name of database and, where practicable, search terms used) EPODOC, FULL TEXT: ENGLISH.

C. Documents considered to be relevant Citation of document, with indication, where appropriate, of the relevant passages X US 2022/0242761 Al (HEINIGER BRUNO et al.) 04/08/2022. 1-15 paragraphs [0001], [0012]- [0013], [0092]- [0093], [0098], [0100],

[0101], [0114], [0118], [0123], and [0126]- [0127]. A NO 2020/0773 Al (NIVA NORWEGIAN INSTITUTE FOR WATER RES) 1-15 03/01/2022. A US 6474264 B1 (GRIMBERG AURELIE et al.) 05/11/2002. 1-15 A CA 3217124 A1 (OHALLORAN JOHN) 03/11/2022. 1-15 [] Further documents are listed in the continuation of Box C * Special categories of cited documents: "P" Document published prior to the filing date but later than the priority date claimed. "A" Document defining the general state of the art which is not considered to be of particular relevance. "T" Document not in conflict with the application but cited to understand the principle or theory underlying the invention. "D" Document cited in the application. " "X" Document of particular relevance; the claimed invention cannot be "E" Earlier application or patent but published on or after the filing considered novel or cannot be considered to involve an essential date. difference when the document is taken alone. "Lt Document which may throw doubt on priority claim(s) or which is "Y" Document of particular relevance; the claimed invention cannot be cited to establish the publication date of another citation or other considered to involve an essential difference when the document is special reason (as specified). combined with one or more other such documents, such combination "0" Document referring to an oral disclosure, use, exhibition, or other being obvious to a person skilled in the art. means. "&" Document member of the same patent family. Danish Patent and Trademark Office Date of completion of the search report Helgeshøj Allé 81 01/10/2024 DK-2630 Taastrup Authorized officer Denmark Erik Lund

Tel.: +45 43 50 80 00 Tel.: +45 43 50 81 40 Page 1 of 4 Version June 2024 g TY DANISH PATENT AND 335 ze — TRADEMARK OFFICE Application No. Search report - patent PA 2024 70076

C. Documents considered to be relevant (continuation) Citation of document, with indication, where appropriate, of the relevant passages Page 2 of 4 Version June 2024 g TY DANISH PATENT AND 335 ze — TRADEMARK OFFICE Application No. Search report - patent PA 2024 70076 Box No. I Certain claims were found unsearchable This search report has not been established in respect of certain claims for the following reasons:

1. O Claims Nos.: because they relate to subject matter not required to be searched. Specifically:

2. [J Claims Nos.: because they relate to parts of the patent application that do not comply with the prescribed requirements to such an extent that no meaningful search can be carried out. Specifically:

3. O Claims Nos.: because of other matters. Specifically: Box No. II Lack of unity of invention was found prior to search Prior to search, multiple independent inventions were found in the patent application. Specifically: Page 3 of 4 Version June 2024 tå Danisk PATENT AND vYVVK mee — TRADEMARK OFFICE Application No.

Search report - patent PA 2024 70076 Supplemental Box Continuation of Box [.] Page 4 of 4 Version June 2024