"kit for preparing a biodegradable putty suitable for promoting underwater coral restoration"

Abstract

Kit for preparing a biodegradable putty suitable for promoting underwater coral restoration, the kit comprising a first component and a second component. The first component comprises at least one radical initiator, the second component comprises at least one accelerator. At least one biodegradable resin is contained in at least one of the first component and the second component.

Description

[0001] “Kit for preparing a biodegradable putty suitable for promoting underwater coral restoration”

[0002] Field of the invention

[0003] This disclosure generally relates to a kit for preparing a biodegradable putty for promoting underwater coral restoration and the relative biodegradable putty. Specifically, the disclosure relates to a biodegradable putty for adhesive bonding at least a portion of a coral on a substrate or to be used as a substrate for coral growth.

[0004] Background

[0005] Coral reefs represent a unique ecosystem characterized by a high degree of biodiversity. Nowadays, coral reefs are threatened by climate changes and sea pollution. For this reason, researchers are trying to save this unique ecosystem using coral restoration techniques. Indeed, corals are grown in specific structures called “nurseries” that are placed either at the bottom or suspended in the sea. Alternative technologies such as the micro-fragmentation technique, wherein fragments of corals coming from the same colony are placed close to each other to improve their growth rate, the Biorock® electrolytic technology (used in mineral accretion in reef restoration, mariculture, and shore protection), or artificial reefs have been utilized. The last step of these technologies, however, always implies transplanting the “new” corals onto the reef.

[0006] Attaching corals somewhere is a practice also diffused in the aquaria, where marine biologists need to fix the corals on a substrate to enhance their growth and health. Document US 2020 / 0053988 Al, for example, discloses mechanical devices wherein a ring, washer, or threaded nut are employed as a propagule mount upon which a coral is set in order to attach, grow and overgrow within a coral nursery system.

[0007] Currently, materials based on synthetic epoxy resins called “putty” are also being used to transplant or attach corals; however, these materials are not explicitly designed for coral attachment but for corrosion protection of boats and oil platforms. These materials, therefore, may be toxic for aquatic organisms and do not biodegrade, bringing serious concerns regarding environmental pollution.

[0008] Marine putties, with more environmentally friendly features, have also been designed, but often, they cannot be mixed underwater and have a slow hardening rate. The same drawbacks were observed for concretes applied underwater using sac-a-Poche to attach the corals to the reef. This concrete can also take hours to harden, and the users should hold the coral, avoiding waves or general sea motions that can alter the position of the placed coral. Other alternatives are wires, cable-ties, coral-clips, and monofilament fishing lines made of synthetic materials that alter the environment and do not ensure biodegradation or suitable stability over time. In addition to the solutions listed above, engineered adhesives to be used in underwater applications are currently available. Document WO 2016 / 196371 Al, for example, discloses a method of adhesively joining segments in non-vertebrate animal structures by using an adhesive aqueous composition comprising a multivalent metal salt and a small organic phosphate compound. Document US 3,657,379 Bl, for example, discloses adhesive systems comprising separated reactive components, specifically a first component comprising a first curable resin combined with a curing agent for a second curable resin and a second component comprising a second curable resin combined with a curing agent for the first curable resin. According to this document, when the two components are combined, the curing agents react with their respective, intercrossed, curable resin achieving strong adhesive bonds. The disclosed adhesive systems may promote bonding on wet surfaces and, as such, may be used underwater. These systems, however, comprise epoxy resins cured by standard epoxy curing, which are not biodegradable and may release plastic and chemical pollutants into the environment.

[0009] Summary of the invention

[0010] The present disclosure provides a kit for preparing a biodegradable, adhesive, and hardening putty particularly effective in promoting underwater coral restoration. The disclosure provides a sustainable, easy, and eco-friendly putty with fast hardening properties and excellent biocompatibility for the corals and the human operators. The kit herein disclosed allows preparing a putty that can be shaped around the corals and - after the shaping is completed - it can harden mimicking a rock. In addition, it can be used as growth substrate for the corals: the growth on the putty allows the corals to reach the coralline rocks and reconstruct as much as possible the original reef ecosystem.

[0011] According to this disclosure, the above object is achieved thanks to the subject-matter to which specific reference is made in the following claims, intended as an integral part of this disclosure. An embodiment of this disclosure provides a kit for preparing a biodegradable putty suitable for promoting underwater coral restoration, in particular for enhance their growth and health. The kit comprises a first component and second component, wherein: i) the first component comprises at least one radical initiator, ii) the second component comprises at least one accelerator, wherein the kit further comprises at least one biodegradable resin contained in in at least one of the first component and the second component.

[0012] When the two components are mixed, the accelerator causes the decomposition of the radical initiator, which creates free radicals and starts the radical polymerization, crosslinking, and hardening of the biodegradable resin.

[0013] The biodegradable resin used for the preparation of the putty herein disclosed is a curable resin. The biodegradable resin may be a resin comprising at least one functional group capable of undergoing radical polymerization. Preferably, the biobased resin comprises at least one acrylate group.

[0014] The biodegradable resin may be selected in the group consisting of: acrylated vegetable oils with at least one acrylate group, preferably epoxidized soybean oil acrylate, epoxidized linseed oil acrylate, epoxidized cardanol oil acrylate; linear, telechelic or star-shaped oligomers of biodegradable polymers, preferably 4-arm poly caprolactone acrylate, 4-arm polylactic acid acrylate, 3 -arm polyethylene glycol; biobased acrylates, preferably lauryl acrylate, isobomyl acrylate, decanediol diacrylate propoxylated glyceryl tri aery lateltaconic acidcontaining molecules, mainly polyesters and polyamides; acrylamide-containing molecules; vinyl esters. Preferably the biobased resin is epoxidized soybean oil acrylate.

[0015] The radical initiator may be selected in the group consisting of organic peroxides, preferably dibenzoyl peroxide, lauroyl peroxide, Tert- butylcumylperoxide, tert-butyl peroxide, 2,5-dimethyl-2,5-di(tert- butylperoxy)hexane, tert-Butyl peroxybenzoate; inorganic peroxides, preferably peroxydisulfate salts, more preferably sodium and ammonium persulfates; azo compounds, preferably azobisisobutyronitrile (AIBN), 2,2’-Azobis(2-(2- imidazolin-2-yl) propane)dihydrochloride (AIBI), 2,2'-Azobis (2,4 dim ethyl valeronitrile) (ABVN).

[0016] In one or more embodiments, the radical initiator is lauroyl peroxide.

[0017] The amount of the radical initiator may be comprised between 0.01% and 10% by weight (w / w) with respect to the total weight of the resin, preferably between 1% and 5% by weight (w / w) of the resin.

[0018] The accelerator may be comprised in the group consisting of amines, preferably para-toluidine ethoxylate, dimethylaniline 2,2'-(4- methylphenylimino)diethanol, priamine, trimethylamine; cobalt-based accelerators, preferably cobalt octoate, cobalt 2-ethylhexanoate.

[0019] The amount of accelerator may be between 0.01% and 10% by weight (w / w) with respect to the total weight of the resin, preferably between 0.5% and 7% by weight (w / w) of the resin.

[0020] The kit may also comprise at least one filler, preferably selected in the group consisting of talc and derivatives, stearic acid and salt derivatives, sepiolite, and clays, hydrophilic fillers, preferably calcium carbonate and derivatives, silica and derivatives; amphiphilic fillers, preferably stearic acid, aleuritic acid, phospholipids; protein-based fillers, preferably zein, silk, keratin; polymeric- based fillers, preferably polyvinylpyrrolidone (PVP), polyhydroxyalkanoates (PHA), poly-P-hydroxybutyrate (PHB), Poly(butylene adipate-co-terephthalate) (PBAT), PLA, PCL, polyvinyl alcohol (PVA), starch, cellulose, polysulfobetaine, poly(N-isopropylacrylamide) (PNIPAM), polyhydroxyethylmethacrylate; conductive fillers, preferably carbon- / boron-based materials, such as graphene, carbon nanotubes, carbon nanofibers, graphite, activated carbon, boron nanotubes, monolayers, crystals; perovskite; natural conductive materials such as cuttlefish ink; conductive polymers such as poly(3,4-ethylenedi oxythiophene) (PEDOT) polystyrene sulfonate (PSS), Polypyrrole (PPy), and combinations of them, silver, gold.

[0021] In one or more embodiments, the filler is selected in the group consisting of zein, calcium stearate, a combination of calcium stearate and sepiolite, graphene.

[0022] The filler may be comprised in the kit in form of powder with a particle size comprised between 1 nm and 10 mm.

[0023] The first component and the second component of the kit may be each housed in a separate container. In one or more embodiments, each component may be housed in a separate packaging or in a separate chamber of a dual chambered packaging. In the case of a kit comprising the two components in a dual -chambered packaging, a separation layer may be used between the two components in order to have each component protected from contact with the other.

[0024] The disclosure also provides a biodegradable putty obtained by:

[0025] - contacting and mixing the first component and the second component of the kit herein disclosed to obtain a putty homogenous solution,

[0026] - allowing the putty homogenous solution to harden.

[0027] The step of allowing the putty homogenous solution to harden may occur in the air or underwater. The biodegradable putty may be used in the restoration of the corals, in particular for use in enhancing their growth and health.

[0028] The disclosure also provides a method for achieving or improving coral restoration by adhesively bonding at least a portion of a coral on a substrate comprising the steps of

[0029] - contacting and mixing the first component with the second component of the kit obtaining a putty homogenous solution,

[0030] - applying the putty homogenous solution on at least one portion of a coral and / or on a substrate,

[0031] -joining the at least one portion of the coral with the substrate,

[0032] - allowing the applied putty homogenous solution to harden.

[0033] The biodegradable putty herein disclosed may be used for adhesively bonding at least a portion of a coral on a substrate and / or may be used as a substrate for coral growth. The biodegradable putty may be used as material for the attachment of corals and, in general, coral reefs, meaning alive corals, the coral tissue, coralline algae, coralline larvae, coral skeleton, coral rubble, coral stones and rocks, and coral sand. These components can be attached all together, but also separately and among them.

[0034] The putty disclosed in the present application is biodegradable. It has a hardening time shorter than other commercial products. The two components of the putty kit do not release any elements in the water, thus showing a great stability. Moreover, the putty can be easily handled underwater and applied on natural substrates such as rocks upon which corals may grow.

[0035] Brief description of the drawings

[0036] The invention will now be described, only by way of example, with reference to the attached figures, in which:

[0037] - Figure 1 schematically shows embodiments of the kit for producing the putty according to the present disclosure;

[0038] - Figure 2 schematically shows a method for preparing a putty according to embodiments of the present disclosure;

[0039] - Figure 3 and Figure 4 show the curing mechanism occurring upon mixing the first component and the second component of the kit; in Figure 3A the epoxidized soybean oil acrylate (ESOA) as biodegradable resins is represented;

[0040] - Figure 5 (panels A and B) shows scanning electron microscope (SEM) images of a putty comprising ESOA as biodegradable resins. Figure 5A is relative to a crosslinked putty free of any filler; Figure 5B shows a crosslinked putty comprising zein as filler;

[0041] - Figure 6 represents FTIR spectra for ESOA, crosslinked ESOA (no fillers), crosslinked Zein-based coral putty, Component 1 (alone), Component 2 (alone), Zein;

[0042] - Figure 7. Interaction of the putty with water. Picture of the two components (components A and B) for a zein-based coral putty of the instant invention (left), a sample of a first commercial putty (in the middle), and a sample of a second commercial putty (right). The arrows indicate the release of ingredients from the putty components;

[0043] - Figure 8. Dynamic mechanical analysis (DMA) measurements. A, B, C, Values of E’ and E” for a sample of a first commercial putty (A), a sample of a second commercial putty (B), and for the putty of the instant disclosure (C, filled with zein in this example), respectively, at 28°C in the function of the time. D, E, F, Values of Tan 5 for the sample of the first commercial putty (D), for the sample of the second commercial putty (E), for the putty herein disclosed (F), respectively, at 28°C in the function of the time;

[0044] - Figure 9. Compression tests. A, Young’s Modulus values for a sample of a first commercial putty (“Comparative A” in the figure), for a sample of a second commercial putty (“Comparative B”), a putty filled with calcium stearate and sepiolite and the initiator lauroyl peroxide at the 5% w / w with respect to the total ESOA (CaSt-Sep Lauroyl 5%), a putty filled with calcium stearate and sepiolite and the initiator lauroyl peroxide at the 1% w / w with respect to the total ESOA (CaSt-Sep Lauroyl 1%), a putty filled with zein and the initiator lauroyl peroxide at the 1% w / w with respect to the total ESOA (Zein Lauroyl 1%). B, Compression strength values for Comparative A, Comparative B, Coral Putty CaSt-Sep Lauroyl 5%, Coral Putty CaSt-Sep Lauroyl 1%, Coral Putty Zein Lauroyl 1%;

[0045] - Figure 10. Biochemical oxygen consumption (mg O2 / IOO mg material) as a function of the time (days) for the putty herein disclosed without filler (Coral Putty no filler), the putty herein disclosed with zein as a filler (Coral Putty Zein filler), a first commercial putty (“Comparative A” in the figure), of a second commercial putty (“Comparative B”), cellulose;

[0046] - Figure 11. Conductive coral putties. A, Picture of a conductive-based coral putty object of the instant disclosure based on graphene. B, C, SEM images of graphene-based conductive coral putty. D, E, Pictures of conductive measurement for air and conductive coral putty; F, Picture of coral nubbins fixed on the conductive coral putty.

[0047] - Figure 12. Fixing corals in aquarium. A, B, Pictures of Stylophora pistillata nubbins fixed with the putty herein disclosed on dowels Fischer. C, D, Pictures of Stylophora pistillata nubbins fixed with the putty herein disclosed on plastic-based substrates;

[0048] - Figure 13. Vertical attachment of the corals in the Maldives. A, B, Pictures of an Acropora sp. fixed with a coral putty comprising calcium stearate and sepiolite on the reef after 0 and 60 days, respectively. The black arrow highlights the growth of the new tissue on the coral putty;

[0049] - Figure 14. Horizontal attachment of the corals in the Maldives. A, B, Pictures of an Acropora sp. fixed with a coral putty comprising calcium stearate and sepiolite on the reef after 0 and 60 days, respectively. The black arrow highlights the growth of the new tissue on the coral putty;

[0050] - Figure 15. Transplanted coral nubbins in the Maldivian sea at day 0, after 6 months, and 1 year by using a putty comprising zein as filler. The arrows indicate the position of the putty;

[0051] - Figure 16. Substrate for coral growth. A, B, C, Fragments of Montipora foliosa attached on a putty comprising calcium stearate and sepiolite after 0, 30 days and 60 days, respectively;

[0052] - Figure 17. Representative schemes of storing modality for the two components of the kit disclosed in the application.

[0053] Detailed description of preferred embodiments

[0054] In the following description, a number of specific details are provided to enable a thorough understanding of embodiments. The embodiments may be practiced without one or more of the specific details or with other methods, components, materials, etc. In other cases, well-known structures, materials or operations are not shown or described in detail to avoid obscuring aspects of the embodiments. Reference throughout this disclosure to “one embodiment” or “an embodiment” means that a particular aspect, structure or feature described in reference to the embodiment is included in at least one embodiment. Thus, the appearances of the phrases “in one embodiment” or “in an embodiment” at various points throughout this disclosure do not necessarily all refer to the same embodiment. Furthermore, the particular aspects, structures or features may be combined in any suitable way in one or more embodiments. The headings provided herein are for convenience only and do not construe the scope or meaning of the embodiments.

[0055] This disclosure provides a kit for preparing a biodegradable, adhesive, and hardening putty particularly effective for use in coral restoration.

[0056] An embodiment of this disclosure provides a kit for preparing a biodegradable putty suitable for promoting underwater coral restoration. The kit comprises a first component and second component, wherein: i) the first component comprises at least one radical initiator, ii) the second component comprises at least one accelerator, wherein the kit further comprises at least one biodegradable resin contained in at least one of the first component and the second component.

[0057] When said first and second components are mixed, the accelerator from the second component causes the decomposition of the radical initiator of said first component, which creates free radicals and starts the radical polymerization, crosslinking, and hardening of the biodegradable resin. The hardening time may vary between 10s and 24-48 hours, preferably between 5 and 15 min. The hardening may occur both in the air and underwater.

[0058] As schematically shown in Figure 1, the two components of the kit are prepared and stored separately from each other and are combined shortly before use to obtain the putty. The kit thus comprises the initiator and the accelerator separated in the two different components: the first component, also named Component A in the examples and figures, and the second component, also named Component B in the examples and in the figures.

[0059] The resin may be present only in the first component together with the initiator (Fig. 1A), only in the second component together with the accelerator (Fig. IB) or, preferably, in both the first component and the second component (Fig. 1C). Panels D-G of Figure 1 schematically show examples wherein different types of resin (indicated as “Resin I”, “Resin II”, “Resin III”) are used. Shown in panel D is the example wherein the first component comprises the initiator with a first resin (Resin I), and the second component comprises the accelerator and a second resin (Resin II); panel E represents a similar combination wherein the two resins are inverted. Panel F and panel G show two embodiments wherein a combination of different resins (for example, Resin I, Resin II, Resin III) is contained in one of the two components, i.e. in Component A together with the initiator (in panel F) or in Component B together with the accelerator (Panel G).

[0060] The biodegradable resin used in the putty herein disclosed is a curable resin.

[0061] The biodegradable resin of the first and second components may be the same.

[0062] The biodegradable resin may be a biobased resin; the term “biobased resin” in the current application refers to any biodegradable naturally derived molecule that can undergo radical polymerization and / or crosslinking (organic molecules, oligomers, polymers). Soybean oil is an example of a biobased molecule derived by extraction from a soya plant.

[0063] The resin used in the putty of the instant disclosure is a biodegradable resin and may be preferably selected among resins widely used in various industrial fields including medicine, food, disposable products, and coating.

[0064] The resin used for the putty of the instant disclosure can undergo radical curing / crosslinking. Other known adhesive systems, for example the one disclosed in document US 3,657,379 Bl may comprise other types of resins, for example epoxidized soybean oil, that are cured not by radical polymerisation but by standard epoxy curing specifically involving the reaction of the epoxy groups with a hardener containing usually amino groups (carboxylic acid or anhydride are also possible). In comparison to the radical polymerization, the epoxy curing occurs in a much longer time period (several hours instead of minutes), especially in a case of an epoxide vegetable oil (like for example the epoxidized soybean oil) which contains much less reactive secondary epoxy groups and not the more reactive primary epoxy groups like in standard synthetic epoxy resins based on bisphenol A diepoxy (DGEBA).

[0065] In one or more embodiments, the biodegradable resin may be selected in the group consisting of acrylated vegetable oils with at least one acrylate group, preferably epoxidized soybean oil acrylate, epoxidized linseed oil acrylate, epoxidized cardanol oil acrylate; linear, telechelic or star-shaped oligomers of biodegradable polymers, preferably 4-arm polycaprolactone acrylate, 4-arm polylactic acid acrylate, 3-arm polyethylene glycol; biobased acrylates, preferably lauryl acrylate, isobornyl acrylate, decanediol diacrylate propoxylated glyceryl tri aery lateltaconic acid-containing molecules, mainly polyesters and polyamides; acrylamide-containing molecules; vinyl esters. Preferably the biodegradable resin is epoxidized soybean oil acrylate.

[0066] In one or more embodiments, the biodegradable resin is a biobased acrylic resin.

[0067] The biodegradable resin comprised in the first component and / or in the second component may be comprised in an amount between 1% and 99% by weight (w / w) with respect to the weight of each component.

[0068] The expression “radical initiator” in the present disclosure refers to any compound that can initiate a radical polymerization.

[0069] The radical initiator may be selected in the group consisting of organic peroxides, preferably dibenzoyl peroxide, lauroyl peroxide, Tert- butylcumylperoxide, tert-butyl peroxide, 2,5-dimethyl-2,5-di(tert- butylperoxy)hexane, tert-Butyl peroxybenzoate; inorganic peroxides, preferably peroxydisulfate salts, more preferably sodium and ammonium persulfates; azo compounds, preferably azobisisobutyronitrile (AIBN), 2,2’-Azobis(2-(2- imidazolin-2-yl) propane)dihydrochloride (AIBI), 2,2'-Azobis (2,4 dim ethyl valeronitrile) (ABVN).

[0070] In one or more embodiments, the radical initiatior is lauroyl peroxide.

[0071] The amount of the radical initiator in the first component - when the reactive resin is comprised in at least one component - may be comprised between 0.01% and 10% by weight (w / w) with respect to the total weight of the resin, preferably between 1% and 5% by weight (w / w) of the resin.

[0072] The amount of the radical initiator in the first component - when the reactive resin is comprised only in the second component - may be comprised between 0.01% and 100% by weight (w / w) with respect to the weight of the component, preferably between 0.01% and 90%, more preferably between 1% and 25% by weight (w / w). The amount of the radical initiator may be between 0.01% and 10% by weight (w / w) with respect to the total weight of the resin (when the resin is contained in at least one of the two components, and the two components are mixed), preferably between 1% and 5% by weight (w / w) of the resin.

[0073] The expression “accelerator” in the present disclosure refers to any compound that can catalyze / speed up the decomposition of the radical initiator. The accelerator may be comprised in the group consisting of amines, preferably para-toluidine ethoxylate, dimethylaniline 2,2'-(4- methylphenylimino)diethanol, priamine, trimethylamine; cobalt-based accelerators, preferably cobalt octoate, cobalt 2-ethylhexanoate.

[0074] The amount of the accelerator in the second component - when the reactive resin is comprised in at least one component - may be comprised between 0.01% and 10% by weight (w / w) with respect to the total weight of the resin, preferably between 0.5% and 7% by weight (w / w) of the resin.

[0075] The amount of accelerator in the second component - when the reactive resin is comprised only in the first component - may be comprised between 0.01% and 100% by weight (w / w) with respect to the weight of said component, preferably between 0.01% and 95% (w / w), more preferably between 0.5% and 30 % by weight (w / w).

[0076] The amount of accelerator may be between 0.01% and 10% by weight (w / w) with respect to the total weight of the resin (when the resin is contained in at least one of the two components, and the two components are mixed), preferably between 0.5% and 7% by weight (w / w) of the resin.

[0077] The first and / or the second component of the putty may also comprise at least one filler.

[0078] The term “filler”, in the present disclosure, refers to additives capable of modifying the chemi cal / phy si cal properties of the putty herein disclosed.

[0079] The filler may be one filler, preferably selected in the group consisting of talc and derivatives, stearic acid and salt derivatives, sepiolite, and clays, hydrophilic fillers, preferably calcium carbonate and derivatives, silica and derivatives; amphiphilic fillers, preferably stearic acid, aleuritic acid, phospholipids; protein-based fillers, preferably zein, silk, keratin; polymeric- based fillers, preferably polyvinylpyrrolidone (PVP), polyhydroxyalkanoates (PHA), poly-P-hydroxybutyrate (PHB), Poly(butylene adipate-co-terephthalate) (PBAT), PLA, PCL, polyvinyl alcohol (PVA), starch, cellulose, polysulfobetaine, poly(N-isopropylacrylamide) (PNIPAM), polyhydroxyethylmethacrylate; conductive fillers, preferably carbon- / boron-based materials, such as graphene, carbon nanotubes, carbon nanofibers, graphite, activated carbon, boron nanotubes, monolayers, crystals; perovskite; natural conductive materials such as cuttlefish ink; conductive polymers such as poly(3,4-ethylenedi oxythiophene) (PEDOT) polystyrene sulfonate (PSS), Polypyrrole (PPy), and combinations of them, silver, gold.

[0080] In one or more embodiments, the filler is selected in the group consisting of zein, calcium stearate, a combination of calcium stearate, and sepiolite, graphene.

[0081] The at least one filler may be comprised in at least one of the two components of the kit, preferably in an amount comprised between 0.01% and 90% by weight (w / w) with respect to the total weight of the resin, preferably between 5% and 60% by weight (w / w) of the resin.

[0082] A putty comprising a conductive filler such as, for example, graphene may present the advantages of concentrating the electric field close to the corals; this promotes a higher deposition concentration of ions such as Ca2+and CCh2' which allows an increase in the growth rate in corals. This is the principle of the system called BioRock®, in which a huge metallic chamber is used to make diffusing the electricity in a specific area. However, also in this case, the corals need to be attached and transplanted on the metallic structure. The conductive paste can be used at the same time to attach the corals to the metallic structure and can guarantee a suitable and more localized electric field in the attached corals. The paste can also be used in other platforms and supports where the current is passing through the paste. This conductive paste can also be used as a component of robots and soft robots that are already being evaluated for coral restoration and underwater purposes.

[0083] The at least one filler may also comprise coral food, molecules that can help restore the coral reefs' environment, such as dimethylsulfide (DMS) and dimethylsulfoniopropionate (DMSP), dimethyl sulfoxide (DMSO), essential elements such as manganese, iron, strontium, and others, compounds and salts source of nitrogen, carbon, and phosphorous, antifouling molecules, antioxidant molecules, antibiotics, antivirus, antiprotozoal, menthol, antifungal molecules, anti-inflammatory, or a combination thereof, to give other functionality to the coral putty and release them to ameliorate the coral state or concentrate the electric field around it.

[0084] The filler may be comprised in the kit putty in the form of powder with a particle size comprised between 1 nm and 10 mm. The filler may be comprised in the kit, preferably in the first component in an amount between 0.001% and 80% by weight (w / w) with respect to the weight of the first component.

[0085] According to a preferred embodiment, the first component of the putty may comprise a biodegradable resin in an amount between 45% and 89% (w / w), at least one filler in an amount between 10% and 50% w / w and a radical initiator in an amount between 1% and 5% (w / w).

[0086] In one or more embodiments, the filler may be present in the second component. When the filler is present in the second component, its amount may vary in an amount between 0.001% and 80% (w / w) with respect to the weight of the component. According to a preferred embodiment, the second component may comprise a biodegradable resin in an amount between 43% and 89.5% (w / w), at least one filler in an amount between 10% and 50% (w / w) and an accelerator in an amount between 0.5% and 7% (w / w).

[0087] The kit for preparing the putty according to the invention is a two- component putty kit. The individual components (first component and second component) may be manufactured and stored separated from each other and combined shortly before use.

[0088] The disclosure also provides a biodegradable putty obtained by:

[0089] - contacting and mixing the first component and the second component of the kit herein disclosed to obtain a putty homogenous solution,

[0090] - allowing the putty homogenous solution to harden.

[0091] The step of allowing the putty homogenous solution to harden may occur in the air or underwater

[0092] The first component of the putty may be prepared by dispersing the radical initiator in the biodegradable resin obtaining a first solution. The resin and the initiator have then to be mixed preferably for 10 seconds at 1200 rpm, 10 seconds at 1400 rpm, 10 seconds at 1600 rpm, 10 seconds at 1800 rpm, and 50 seconds at 2000 rpm. Said mixing step should be repeated until the solution becomes homogeneous (preferably two cycles of mixing).

[0093] At this point, it is possible to add, if required, a filler to the solution. If the filler is added, the solution has to be mixed again at the same conditions as the previous mixing step, but this time preferably for three cycles of mixing.

[0094] The second component is produced by employing the same procedure used for the first component thus by dispersing the accelerator in the biodegradable resin obtaining a second solution.

[0095] The different ingredients of the two components can be mixed using different techniques or tools such as a homogenizer, mixer, blender, extruder, sonicator, and general tool to make emulsions.

[0096] The first and the second component may be mixed at the ratio first component on the second component comprised between 1 :30 to 1 :0.3 (w / w), preferably between 1 :0.5 to 1 :5 (w / w).

[0097] After the mixing step, the putty shows a crosslinked material wherein the fillers are fully dispersed inside the matrix.

[0098] Said first and second components, when stored separately, have each a consistency of a viscous liquid or a paste; they are stable and do not harden over time. When said first and second components are mixed, the accelerator of said second component causes the decomposition of said radical initiator of said first component, which in turn creates free radicals and starts the radical polymerization, crosslinking, and hardening of the biodegradable resin.

[0099] The hardening time may vary between 10 seconds and 48 hours, preferably between 10 seconds and 24 hours, more preferably between 5 min and 15 min.

[0100] The hardening of the biodegradable resin may occur both in the air and underwater. The kit may be used in the restoration of a coral for adhesively bonding at least a portion of a coral on a substrate. The kit may be used in the restoration of a coral as a substrate for coral growth.

[0101] One or more embodiments of the present application provides a method for achieving or improving coral restoration by adhesively bonding at least a portion of a coral on a substrate comprising the steps of:

[0102] - applying the first component and the second component of the putty kit on at least a portion of a coral and / or on the substrate,

[0103] -joining the at least a portion of a coral with the substrate,

[0104] - allowing the applied putty to harden.

[0105] Preferably, a step of mixing the first component and the second component is carried out before the applying step. More preferably, the first and the second component may be mixed at the ratio first component on the second component comprised between 1 :30 to 1 :0.3 (w / w), preferably between 1 :0.5 to 1 :5 (w / w) before the applying step.

[0106] The step of allowing the applied putty to harden may be carried out for a period of time comprised of 10 seconds and 48 hours, preferably between 10 seconds and 24 hours, more preferably between 5 min and 25 min.

[0107] The putty of the instant application suitable for adhesively bonding at least a portion of a coral wherein the expression “at least one portion of a coral” refers to, in general, coral reefs, meaning alive corals, the coral tissue, coralline algae, coralline larvae, coral skeleton, coral rubble, coral stones and rocks, and coral sand. These components can be attached all together, but also separately and among them. The biodegradable putty may be used in the restoration of the coral for adhesively bonding at least a portion of a coral on a substrate. The biodegradable putty may be used in the restoration of the coral as a substrate for coral growth.

[0108] The two components of the kit can be used and stored, keeping them separated and mixed at the operating moment, or used and stored in a unique formulation by using a separator / isolator / insulator between the two components.

[0109] Each component of the kit may be housed in a separate packaging or in a separate chamber of dual -chambered packaging. In the case of a kit comprising the two components in a dual-chambered packaging, a separation layer may be used between the two components in order to have each component protected from contact with the other. The separation layer may be selected in the group consisting of: i) a film / layer / coating from a water soluble, biodegradable polymer like for example alginate, PVA, starch; ii) a film / layer / coating from a waterinsoluble biodegradable polymer like PCL, PLA; iii) a film / layer / coating of pure filler selected between the filler disclosed for the putty (already present in the formulation or new). The putty disclosed in the instant application has a hardening time shorten than other commercial putty products, and the hardening time may be regulated by changing the type of resin or filler, the quantities of initiator and accelerator. The two separated components do not release any elements in the water, showing great stability.

[0110] EXAMPLES

[0111] Example 1. Preparation of the biodegradable putty

[0112] Example 1 discloses the preparation of a putty by using a kit wherein the resin is contained in both the two components, as schematically shown in Figure 1C. The presence of the resin in both the components has the advantage to make easier the measure of the right proportions of the ingredients as well as their mixing.

[0113] The first component is produced by dispersing the radical initiator lauroyl peroxide at 1.5% by weight (w / w) with respect to the total weight of the total amount of epoxidized soybean oil acrylate (ESOA) (contained in the two components, also named “Component A” and “Component B” in the present disclosure) and obtaining a first solution.

[0114] The ESOA and the initiator were mixed using a THINKY Mixer (https: / / www.thinkymixer.com / en-us / ) running preferably for 10 seconds at 1200 rpm, 10 seconds at 1400 rpm, 10 seconds at 1600 rpm, 10 seconds at 1800 rpm, and 50 seconds at 2000 rpm. Once the first solution becomes homogenous (preferably, two cycles of mixing), the filler can be added and mixed again (preferably, three cycles of mixing).

[0115] The amount of filler used depends on the filler itself and can be adapted in function of their features and miscibility with the oil.

[0116] For the instant example, the filler comprises zein, calcium stearate, a combination of calcium stearate and sepiolite (in a weight ratio 2.6: 1) and graphene.

[0117] The weight of the filler with respect to the weight of EOSA was: 50% (w / w) for zein and graphene, and 54% (w / w) for both the calcium stearate alone and the combination calcium stearate with sepiolite.

[0118] The second component (Component B) is produced by mixing 1.5% (w / w) of the accelerator para-toluidine ethoxylate by weight (w / w) with respect to the total weight of ESOA in the final coral putty (of the first component and the second component).

[0119] The method for preparing the putty is schematically presented in Figure 2.

[0120] When the first component (A) and the second component (B) are mixed, the radical reaction is triggered, as also shown in Figures 3 and 4. After the crosslinking following the mixing of the two components, the putty matrix is smooth and compact if the filler is not used, as shown in Figure 5A. In contrast, the putty matrix shown in Figure 5B shows a homogenous distribution of the dispersed filler, zein in the specific example.

[0121] To confirm the occurred crosslinking, attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy was used. The spectra of EOSA alone, ESOA crosslinked without filler, crosslinked zein-based coral putty, component A alone, component B alone, and zein alone are reported in Figure 6.

[0122] ESOA spectrum showed the following vibrations modes: a weak O-H stretching mode at 3508 cm’1of the small amount of hydroxyl group in the later side of the chains; asymmetric and symmetric CEE and CEE stretching modes at 2955, 2926, 2870, and 2855 cm’1connected with the aliphatic chain; C=O stretching mode of the esters between the fatty acid and the glycerol at 1740 cm’1; C=O stretching mode of the acrylic ester in the chain at 1722 cm’1; acrylic C=C stretching modes of the acrylic group (trans and cis isomers) at 1636 and 1618 cm’J; CH2 and CH3 bending modes at 1465 and 1373 cm’1, respectively; C-O-C stretching modes for the acrylic ester and the fatty ester at 1188 and 1157 cm’1, respectively; wagging terminal CH2 of the acrylic group mode at 809 cm’1; rocking CH3 of the fatty chains mode at 723 cm’1.

[0123] Changes connected with the crosslinking process can be observed in the spectrum of the crosslinked matrix without zein as filler. To highlight these changes, the two spectra were normalized in the function of the rocking CH3 of the fatty chain mode at 723 cm’1, which being the terminal part of the fatty chains, is not involved in the reaction (see Figures 3 and 6). An intense reduction of the signals of the acrylic group at 1636, 1618, and 809 cm’1compared to the ESOA sample was observed. The C=O directly connected to the acrylic group was much less intense, and now it cannot be distinguished from the C=O of the fatty acid, suggesting a potential shift at higher wavenumbers due to the lack of the reacted acrylic group. Indeed, the peak is centered at 1728 cm’1. Likewise, the C-O-C signal at 1188 cm’1drastically reduced its intensity with respect to the ESOA spectrum, now less intense than the C-O-C stretching mode of the fatty acid chain. These changes confirm the occurring crosslinking reaction, which directly involves the acrylic group, as schematized in Figures 3 and 4.

[0124] The coral putty comprising the crosslinked matrix with the zein as filler presents an overlapping of the peaks of ESOA and zein. Zein peaks cover the acrylic doublet, but the other signals of the occurred crosslinking can be observed. Indeed, the C=O stretching modes are overlapped and centered at 1728 cm’1, the C-O-C of the fatty acid chain is more intense than the ones of the acrylic ester, and the wagging terminal CH2 of the acrylic group is less intense than the rocking CH3 of the fatty chain mode. This is also confirmed in the spectra of components A and B, where these peaks are similar to the pristine ESOA. Also, here, the typical peaks of zein can be recognized. Specifically, zein vibration modes are: N- H stretching occurs at 3288 cm’1; O-H stretching at 3201 cm’1; the typical C-H aromatic and aliphatic stretching vibration from 3090 to 2850 cm’1, which confirms the presence of aromatic and aliphatic groups of the zein aminoacid; the Amide I centered at 1646 cm’1, Amide II at 1516 cm’1, and Amide III at 1446 cm’1characteristic of the peptide bond; and C-N stretching at 1235 cm’1.

[0125] Thus, when components 1 and 2 are mixed, the radical reaction is triggered, as shown in Figures 3 and 4. The dibenzoyl peroxide, thanks to the tertiary amine, starts propagating the free radical, which reacts with the acrylate group generating more free radicals and crosslinking the ESOA matrix. The zein passively acts as a filler.

[0126] Example 2, Water stability

[0127] The performances of the putty herein disclosed were compared with samples of other two commercial bi-components putties used in aquaria and underwater applications; the samples of the two commercial products (lamSub and Aquamend) are identified in the figures and in the following examples as “Comparative A” and “Comparative B” respectively. Here, the two components labeled A and B of the three different putties were immersed in salty water. As can be noticed in Figure 7, in both the comparative samples component A released ingredients from the matrix and this may suggest a potential toxicity for the environment, while in the coral putty of the instant application (in this example filled with zein), both the kit components were macroscopically stable during the time in salty water. The putty herein disclosed differs from the other sample of the two commercial bi-components products for being biobased, biodegradable, based on radical polymerization not on epoxy curing, has a faster hardening time, a better water stability and it is nontoxic. In the scenario of sub-applying the coral putty for coral transplantation, this stability and the no release of any potentially toxic element is a crucial point for developing more eco-friendly hardening materials for this purpose.

[0128] Example 3, Hardening rate

[0129] Another crucial point for a successful coral transplantation is the hardening time after the mixing of the two components of the putty kit. The hardening time should be slow enough to give the time to the operator / user / submarine to shape the paste and the coral inside it, but on the other hand, fast enough to avoid that the system comprising the putty, the coral and the rock can be altered due to the softness of the paste. The coral can fall out from the paste due to its weight, the water current, and the animals if it is not well-entrapped.

[0130] For this reason, the hardening rates of the putty herein disclosed (in this example filled with zein), of samples of the two comparative commercial products were tested by using DMA and monitoring the variation of Storage and Loss Modulus (E’ and E”, respectively) and Tan 5 during the time and at a fixed temperature of 28°C, as shown in Figure 8. This temperature is present in the sea / ocean in the tropical area where corals live.

[0131] The DMA results for are reported in Figure 8. As can be noticed, a quick decrease of the loss modulus and an increase of the E’ were recorded for the coral putty herein disclosed suggesting a stable hardening after 25-30 minutes (panel C). On the other hand, the comparative commercial products (panel A and panel B) were not hard after 80 and 180 minutes, respectively. Similarly, the value of Tan 5 quickly decreased for the putty of the instant invention (panel F), while for the two comparative commercial products (panel D and panel E), it remained constant along the measurements.

[0132] Example 4, Compression Test

[0133] The compression tests were performed to compare the mechanical properties of crosslinked putty herein disclosed with samples of the two commercial products. Putties are filled with calcium stearate and sepiolite and zein with lauroyl peroxide used as initiator. The results of the compression tests are shown in Figures 9A and 9B. Young’s Modulus values are obtained from the stress-strain curves for the Comparative A and the Comparative B commercial product samples, and coral putties herein disclosed with different compositions as reported in Figure 9A. Similarly, the compression strength results for the two commercial products, and for the coral putties herein disclosed are displayed in Figure 9B. Considering the Young’s Modulus and the compression strength, the coral putties showed comparable mechanical properties to the epoxy resin-based materials commercially available. Interestingly, by changing the quantity of initiator, the properties of the coral putties were not significantly modified, suggesting that for these fillers, the 1% w / w of lauroyl with respect to total ESOA was enough to achieve performance comparable to the commercial putties. Noteworthy is that the values of compression strength of above lOMPa are comparable to the typical concrete and bricks used for building (Abdalla et al. (2019) Results in Materials; 1, 100004; Parashar et al. (2012) International Journal of Scientific and Research Publications, Volume 2, Issue 7). These results confirm the ductility of the putty of the instant application.

[0134] Example 5, Biodegradation

[0135] Biodegradation of the putty of the instant invention free of filler (Coral Putty no filler (ESOA oil + radical initiator + radical accelerator, without fillers), the putty herein disclosed comprising zein as filler (Coral Putty Zein Filler), commercial product (Comparative A), commercial product (Comparative B), and cellulose (Cellulose) were evaluated by Biochemical Oxygen Demand (BOD) test in salty water. The results are reported in Figure 10. Both the “unfilled” putty and the one filled with zein are biodegradable and well -recognized by bacteria. The same occurred to the cellulose used as the control sample. However, the two comparative commercial products were not biodedagrable.

[0136] Example 6, Conductive putty

[0137] Another diffused way for speeding up the growth of corals is the use of electric field which can enhance the accumulation / deposition of calcium on the corals. This technology is called Biorock electrolytic technology, and the phenomenon is correlated with an increased fixation of calcium by corals in their skeleton and thus improving their growth rate.

[0138] The putty disclosed in the instant application can be functionalized with conductive fillers in order (not only to be used as the other coral putties as hardening materials) but also to localize and spread the electric field close to the corals. Conductive coral putties can be prepared by using fillers such as carb on / b or on-based materials, specifically graphene, carbon nanotubes, carbon nanofibers, graphite, activated carbon, boron nanotubes, monolayers, crystals; perovskite; natural conductive materials such as cuttlefish ink; conductive polymers such as poly(3,4-ethylenedi oxythiophene) (PEDOT) polystyrene sulfonate (PSS), Polypyrrole (PPy), and combinations of them, silver, gold.

[0139] In Figure 11 A, a conductive coral putty loaded with graphene powder is shown. The flakes of the carbon material were well -dispersed inside the oily matrix, as can be observed from the SEM images reported in Figures 11B and 11C. Indeed, the surface and the cross-section resulted in being well homogenous, and by zooming at higher magnification, it was possible to observe the good connection among the graphene flakes inside the putty. The coral putty was capable of hardening, performing as the other coral putty. At the same time, the material has excellent conductive properties.

[0140] Placing the electrodes of a tester in the air, no resistance was measured, while on the surface of coral putty, it registered a low resistance for the electrons and, thus, a good conductivity (see Figures 1 ID and 1 IE).

[0141] Finally, this conductive coral putty was biocompatible for corals, not creating any reaction on the animals in the short and long term, as can be noticed in Figure 1 IF.

[0142] Example 7, Application of the putty on corals

[0143] Different formulations of putties according to embodiments herein disclosed were applied on corals of different species such as Stylophora pistillata, Acropora sp. etc. Tests of biocompatibility and capacity of the putties to mechanically support the corals and their growth, were carried out first at aquaria level and then in the open field.

[0144] In the aquarium, the coral putty (in this example filled with zein) was utilized to fix the corals on different supports and different surfaces, as shown in Figures 12A, 12B, 12C, and 12D. No signal of stress was noticed, and the coral can normally growth vertically but also on the surface of the material.

[0145] Afterward, the coral putties were applied in the Maldivian sea to out-plant corals on the reef at the dept of 10 meters and for a period between 2 months and 1 year. The first parameters monitored for the putties were their capacity to adhere on the rocks (natural substrate) and mechanically support the corals along the time. The coral putties ensure a strong and quick adhesion to the natural substrate. The excellent performance of the coral putties (in Figures 13 and 14 the coral putties are filled with calcium stearate and sepiolite, while in Figure 15 the coral putty is filled with zein) allowed the adhesion of corals not only vertically attached but also horizontally, confirming the quick and strong adhesive properties of those materials. Usually, other epoxy resins do not allow horizontal adhesion of corals due to their slow hardening and, thus, the lack of proper mechanical properties during the out-plantation.

[0146] As observed in aquaria, also in nature and in the real environment, corals were healthy and growth vertically and on the surface of the putties. This is a crucial point first because the coral recognized the putties as a good substrate where it can grow; second, while the putty is slowly biodegrading, at the same time the coral tissue can reach the rock and fix itself on the reef incorporating the coral putties. These features are crucial for a successful and ecofriendly out- plantation. Finally, the coral putty can be used as the substrate for growth of the corals also using growing techniques such as coral fragmentation (as shown in Figure 16).

[0147] Example 8, Putty kit packaging and storing

[0148] The putty kit components of the present disclosure may be used, stored, or sold keeping the two components separated and mixed at the operating moment.

[0149] In one or more embodiments, the components of the putty kit may be stored in a unique formulation by using a separator layer between the two components. The separator may be a film from a water soluble, biodegradable polymer like alginate, PVA, PVP, or starch, for example. Alternatively, film made of a water- insoluble biodegradable polymer like PCL, PLA, PHA, PHB, PBAT, PBS, and / or coatings / layers of pure filler (already present in the formulation or new) can be utilized to prevent the touching of both components. Figure 17 schematically reports examples of packaging of the putty herein disclosed. These results provide a putty kit and a biodegradable putty for use in underwater coral restoration. In particular, the kit and the biodegradable putty are provided for use in enhancing the coral growth and health. Preferably, the kit and the biodegradable putty are provided for use in adhesively bonding at least a portion of a coral on a substrate and / or for use as a substrate for coral growth. Altogether the results show that the putty herein disclosed can be easily handled underwater and applied on natural substrates such as rocks; it can be used as a substrate upon which the coral can grow. In addition, it can be easily produced; does not require any organic solvents for its manufacturing; it is biodegradable in a proper time scale; it is biodegradable and free from toxic compounds; its hardening is fast and controllable in the air and underwater; the corals are easily attached to the rocks underwater and can grow after transplantation.

Claims

CLAIMS1. Kit for preparing a biodegradable putty suitable for promoting underwater coral restoration, the kit comprising a first component and a second component, wherein: i) the first component comprises at least one radical initiator, ii) the second component comprises at least one accelerator, wherein the kit further comprises at least one biodegradable resin contained in at least one of the first component and the second component, wherein the biodegradable resin is epoxidized soybean oil acrylate.

2. Kit according to claim 1, wherein the biodegradable resin is contained in at least one between the first component and the second component in an amount between 1% and 99% by weight (w / w) with respect to the total weight of said components.

3. Kit according to claim 1 or claim 2, wherein radical initiator is selected in the group consisting of organic peroxides, preferably dibenzoyl peroxide, lauroyl peroxide, Tert-butylcumylperoxide, tert-butyl peroxide, 2,5-dimethyl-2,5-di(tert- butylperoxy)hexane, tert-Butyl peroxybenzoate; inorganic peroxides, preferably peroxydisulfate salts, more preferably sodium and ammonium persulfates; azo compounds, preferably azobisisobutyronitrile (AIBN), 2,2’-Azobis(2-(2- imidazolin-2-yl) propane)dihydrochloride (AIBI), 2,2'-Azobis (2,4 dim ethyl valeronitrile) (ABVN).

4. Kit according to any one of the preceding claims, wherein the resin is contained in at least the first component and the amount of the radical initiator is comprised between 0.01% and 10% by weight (w / w) with respect to the weight of said first component, preferably between 1% and 5% (w / w).

5. Kit according to any one of the preceding claims, wherein the accelerator is selected in the group consisting of amines, preferably para-toluidine ethoxylate, dimethylaniline 2,2'-(4-methylphenylimino)diethanol, priamine, trimethylamine; cobalt-based accelerators, preferably cobalt octoate, cobalt 2-ethylhexanoate.

6. Kit according to any one of the preceding claims, wherein the resin is contained in at least the second component and the amount of the at least one accelerator is comprised between 0.01% and 10% by weight (w / w) with respect to the weight of the second component, preferably between 0.5% and 7% (w / w).

7. Kit according to any one of the preceding claims, further comprising atleast one filler, preferably selected in the group consisting of talc and derivatives, stearic acid and salt derivatives, sepiolite, and clays, hydrophilic fillers, preferably calcium carbonate and derivatives, silica and derivatives; amphiphilic fillers, preferably stearic acid, aleuritic acid, phospholipids; protein-based fillers, preferably zein, silk, keratin; polymeric-based fillers, preferably polyvinylpyrrolidone (PVP), polyhydroxyalkanoates (PHA), poly-P- hydroxybutyrate (PHB), Poly(butylene adipate-co-terephthalate) (PBAT), PLA, PCL, polyvinyl alcohol (PVA), starch, cellulose, polysulfobetaine, poly(N- isopropylacrylamide) (PNIPAM), polyhydroxyethylmethacrylate; conductive fillers, preferably carbon- / boron-based materials, such as graphene, carbon nanotubes, carbon nanofibers, graphite, activated carbon, boron nanotubes, monolayers, crystals; perovskite; natural conductive materials such as cuttlefish ink; conductive polymers such as poly(3,4-ethylenedi oxythiophene) (PEDOT) polystyrene sulfonate (PSS), Polypyrrole (PPy), and combinations of them, silver, gold.

8. Kit according to claim 7, wherein the filler is comprised in an amount between 0.01% and 90% by weight (w / w), preferably between 5% and 60%, with respect to the weight of the biodegradable resin.

9. Biodegradable putty suitable for promoting underwater coral restoration obtained by:- contacting and mixing the first component and the second component of the kit according to any one of claims 1 to 8 to obtain a putty homogenous solution,- allowing the putty homogenous solution to harden.

10. Method for improving coral restoration by adhesively bonding at least a portion of a coral on a substrate comprising the steps of- contacting and mixing the first component with the second component of the kit according to any one of claims 1 to 8 and obtaining a putty homogenous solution,- applying the putty homogenous solution on at least one portion of the coral and / or on a substrate,-joining the at least one portion of the coral with the substrate,- allowing the applied putty homogenous solution to harden.

11. Method according to claim 10, wherein the step of allowing the applied putty homogenous solution to harden may be carried out for a time periodcomprised between 10 seconds and 48 hours, preferably between 10 seconds and 24 hours, more preferably between 5 min and 15 min.

12. Use of the biodegradable putty according to claim 9, for adhesively bonding at least a portion of a coral on a substrate.

13. Use of the biodegradable putty according to claim 9, as a substrate for coral growth.

Images