Development of Antifouling Strategies for Marine Applications

Marine biofouling is an undeniable challenge for aquatic systems since it is responsible for several environmental and ecological problems and economic losses. Several strategies have been developed to mitigate fouling-related issues in marine environments, including developing marine coatings using nanotechnology and biomimetic models, and incorporating natural compounds, peptides, bacteriophages, or specific enzymes on surfaces. The advantages and limitations of these strategies are discussed in this review, and the development of novel surfaces and coatings is highlighted. The performance of these novel antibiofilm coatings is currently tested by in vitro experiments, which should try to mimic real conditions in the best way, and/or by in situ tests through the immersion of surfaces in marine environments. Both forms present their advantages and limitations, and these factors should be considered when the performance of a novel marine coating requires evaluation and validation. Despite all the advances and improvements against marine biofouling, progress toward an ideal operational strategy has been slow given the increasingly demanding regulatory requirements. Recent developments in self-polishing copolymers and fouling-release coatings have yielded promising results which set the basis for the development of more efficient and eco-friendly antifouling strategies.


Introduction
Marine biofilm development is a complex and dynamic process comprising several organisms and interactions, which can be affected by different factors, from surface properties to environmental parameters and microbial content [1][2][3][4]. Indeed, biofilms are a common feature on all aquatic submerged surfaces, contributing to marine biofouling, which is responsible for several detrimental impacts on shipping efficiency, aquaculture industries, equipment corrosion, and maintenance, as well as disturbances in ecosystems [5][6][7]. Since cell adhesion and biofilm formation are primordial steps to macrofouling, the most promising marine biofouling mitigation approach is delaying and controlling microfouling events [8,9].
Even though the schematic conceptual biofilm developmental model based on five stages (reversible attachment of planktonic cells, irreversible attachment, biofilm maturation by the development of microcolonies and high extracellular polymeric substance (EPS) production, maturation of the biofilm, and dispersal/detachment) has been widely generalized to describe all biofilms [10], this model does not necessarily describe the complexity of biofilms in the real world, including industrial, clinical, and natural settings as marine environments. Indeed, this model was recently reviewed by the scientific community, which proposed a most inclusive model involving three major events: aggregation, growth, and disaggregation [11]. Therefore, although no developmental model accurately represents biofilm formation for all microorganisms, numerous in vitro systems have been designed to study biofilm formation and development to better mimic real conditions [12,13]. Moreover, some of these in vitro studies are posteriorly validated and/or confirmed by in situ studies in real marine environments [14]. The advantages and limitations of both study types must be considered when choosing the most appropriate method.
There is a pressing need to develop novel antibiofilm surfaces to manage concerns associated with marine fouling and comply with the increasingly strict and demanding legislation in this area [15,16]. Some of these policies involve banning biocides or antifouling paints due to their high persistence and toxicity on non-target marine organisms [17], as well as providing guidelines for the control and management of ship biofouling to minimize the transfer of invasive aquatic species [18]. Several marine coatings have been developed and tested under in vitro and/or in situ assays. Advancements in polymer science, nanotechnology, and the progress of innovative surface models inspired by nature are expected to significantly impact the improvement of antifouling methodologies, contributing to the development of a new generation of environmentally friendly marine coatings.
This review aims to briefly collect evidence on the development and concerns of marine biofouling and introduce a brief overview of the current marine antifouling strategies used. The advancement and the impact of different marine coatings on marine biofilm development are addressed, focusing on the importance, advantages, and limitations of in vitro and in situ studies.

Marine Biofouling
Marine biofouling is a dynamic natural process that comprises both microfouling and macrofouling events. Although the diversity and prevalence of fouling organisms depend on geographic location, seasonal variations, and different interactions [19], microfouling includes forming a conditioning film over the submerged surface, the adhesion of microfouler organisms (mainly bacteria, cyanobacteria, and diatoms), followed by biofilm development. In turn, macrofouling implies the attachment and settlement of soft fouler organisms, such as algae, corals, sponges, anemones, tunicates, hydroids, and additional marine invertebrates (e.g., larvae of brine shrimp), as well as barnacles, mussels, bryozoans, and tubeworms (hard fouler organisms) ( Figure 1) [19,20].
Microorganisms 2022, 10, x FOR PEER REVIEW 2 numerous in vitro systems have been designed to study biofilm formation development to better mimic real conditions [12,13]. Moreover, some of these in studies are posteriorly validated and/or confirmed by in situ studies in real m environments [14]. The advantages and limitations of both study types mu considered when choosing the most appropriate method. There is a pressing need to develop novel antibiofilm surfaces to manage con associated with marine fouling and comply with the increasingly strict and deman legislation in this area [15,16]. Some of these policies involve banning biocid antifouling paints due to their high persistence and toxicity on non-target m organisms [17], as well as providing guidelines for the control and management of biofouling to minimize the transfer of invasive aquatic species [18]. Several m coatings have been developed and tested under in vitro and/or in situ as Advancements in polymer science, nanotechnology, and the progress of innov surface models inspired by nature are expected to significantly impact the improve of antifouling methodologies, contributing to the development of a new generati environmentally friendly marine coatings.
This review aims to briefly collect evidence on the development and concer marine biofouling and introduce a brief overview of the current marine antifo strategies used. The advancement and the impact of different marine coatings on m biofilm development are addressed, focusing on the importance, advantages, limitations of in vitro and in situ studies.

Marine Biofouling
Marine biofouling is a dynamic natural process that comprises both microfoulin macrofouling events. Although the diversity and prevalence of fouling organisms de on geographic location, seasonal variations, and different interactions [19], microfo includes forming a conditioning film over the submerged surface, the adhesio microfouler organisms (mainly bacteria, cyanobacteria, and diatoms), followed by bi development. In turn, macrofouling implies the attachment and settlement of soft f organisms, such as algae, corals, sponges, anemones, tunicates, hydroids, and addit marine invertebrates (e.g., larvae of brine shrimp), as well as barnacles, mu bryozoans, and tubeworms (hard fouler organisms) ( Figure 1) [19,20].  Representation of the marine biofouling process and the main parameters/factors that affect microfouling and macrofouling events. Microfouler organisms include mainly marine bacteria, cyanobacteria, and diatoms, while macrofouler organisms comprise algae, corals, sponges, anemones, tunicates, hydroids, and additional marine invertebrates (soft macrofouler organisms), as well as barnacles, mussels, bryozoans, and tuberworms (hard macrofouler organisms). This image was created with the software BioRender (https://biorender.com/).

Marine Antifouling Strategies
Several strategies have been used to mitigate the effects of marine biofouling. These approaches can prevent and/or delay biofilm development and the attachment of macrofoulers, comprising antimicrobial, antibiofilm, and antifouling surfaces [41], or control already established biofilms and fouling communities ( Figure 3, Table 1). Control methodologies involve using bacteriophages, enzymes, QS inhibitors, disinfectants, additional treatment methods, and cleaning technologies [38,[42][43][44][45] (Figure 3). A range of criteria should be evaluated to select the most suitable marine antifouling strategy, including effectiveness, safety, biosecurity, compatibility with the materials of devices/equipment, and feasibility. First, effectiveness implies evaluating the activity, concentration, or intensity spectrum of antifouling activity and required exposure time. The antifouling strategy must be safe for the environment (ecotoxicological safety) and operators, as well as not exacerbate the biosecurity risk of releasing and establishing nonindigenous species. Moreover, the antifouling strategy should be compatible with the Figure 2. Main consequences of marine biofouling. This graphic representation shows the major effects of marine biofouling on submerged devices/equipment, such as sensors, buoys, cameras, aquaculture facilities, ships, and oil and gas platforms. This image was created with the software BioRender (https://biorender.com/).

Marine Antifouling Strategies
Several strategies have been used to mitigate the effects of marine biofouling. These approaches can prevent and/or delay biofilm development and the attachment of macrofoulers, comprising antimicrobial, antibiofilm, and antifouling surfaces [41], or control already established biofilms and fouling communities ( Figure 3, Table 1). Control methodologies involve using bacteriophages, enzymes, QS inhibitors, disinfectants, additional treatment methods, and cleaning technologies [38,[42][43][44][45] (Figure 3). A range of criteria should be evaluated to select the most suitable marine antifouling strategy, including effectiveness, safety, biosecurity, compatibility with the materials of devices/equipment, and feasibility. First, effectiveness implies evaluating the activity, concentration, or intensity spectrum of antifouling activity and required exposure time. The antifouling strategy must be safe for the environment (ecotoxicological safety) and operators, as well as not exacerbate the biosecurity risk of releasing and establishing non-indigenous species. Moreover, the antifouling strategy should be compatible with the equipment itself to avoid damaging systems or other components of the devices/equipment. It should also be cost-effective and fulfill infrastructure requirements [38]. equipment itself to avoid damaging systems or other components of the devices/equipment. It should also be cost-effective and fulfill infrastructure requirements [38]. Antifouling paints containing arsenic, zinc, tin, and mercury were commonly used as the initial strategy to deal with marine biofouling [46,47] until their toxicity on the surrounding marine environment was demonstrated [48][49][50]. Indeed, in the 1960s, coatings incorporating a tributyl tin (TBT)-based biocide were the first to present robust effectiveness with a relatively low production cost. However, several findings indicated the negative impacts of TBT-based compounds related to their persistence and toxicity, showing adverse effects on non-target marine organisms. Several governments restricted its use, and the International Maritime Organization decided to ban the use of this type of biocide in the manufacturing of antifouling paints in 2003 and the presence of these paints on ship surfaces from 2008 [17].
Therefore, further biofouling treatments have been applied, including thermal stress, osmotic shock, deoxygenation, UV and laser radiation, and hydrodynamic and acoustic cavitation [38,44,45,51]. The most commonly available cleaning technologies are brushing, scraping, pressure cleaning with water/air jetting, or mechanical cleaning using wipers [33,38,44,45,51,52]. These mitigation strategies vary in their effectiveness in removing biofouling organisms and in their suitability for use on different marine surfaces. For instance, although the intensity of cavitation erosion of submerged surfaces depends on the material properties of the surface, liquid temperature, and the distance from the edge of the working tool to the fouling which should be removed, cavitation technology allows Antifouling paints containing arsenic, zinc, tin, and mercury were commonly used as the initial strategy to deal with marine biofouling [46,47] until their toxicity on the surrounding marine environment was demonstrated [48][49][50]. Indeed, in the 1960s, coatings incorporating a tributyl tin (TBT)-based biocide were the first to present robust effectiveness with a relatively low production cost. However, several findings indicated the negative impacts of TBT-based compounds related to their persistence and toxicity, showing adverse effects on non-target marine organisms. Several governments restricted its use, and the International Maritime Organization decided to ban the use of this type of biocide in the manufacturing of antifouling paints in 2003 and the presence of these paints on ship surfaces from 2008 [17].
Therefore, further biofouling treatments have been applied, including thermal stress, osmotic shock, deoxygenation, UV and laser radiation, and hydrodynamic and acoustic cavitation [38,44,45,51]. The most commonly available cleaning technologies are brushing, scraping, pressure cleaning with water/air jetting, or mechanical cleaning using wipers [33,38,44,45,51,52]. These mitigation strategies vary in their effectiveness in removing biofouling organisms and in their suitability for use on different marine surfaces. For instance, although the intensity of cavitation erosion of submerged surfaces depends on the material properties of the surface, liquid temperature, and the distance from the edge of the working tool to the fouling which should be removed, cavitation technology allows lower surface damage compared to brush-based technologies [53]. Moreover, nowadays, the cleaning of boats, ships, and additional moveable marine equipment such as cages and nets can be performed in a dry-dock or by in-water cleaning technologies [44,53]. Although in-water biofouling approaches can be cheaper than onshore activities, they may present higher chemical contamination and biosecurity risks, e.g., the application of underwater technology may increase the recolonization of surrounding surfaces [54].
Enzymes have also been proposed as an alternative to traditional antifouling compounds since they can act on the breakdown of adhesive components and the catalytic production of repellent compounds in situ [42]. A broad spectrum of aquatic disinfectants, such as Triple7 Enviroscale Plus ® (citric acid: 30-60%; lactic acid: 30-60%), Descalex ® (sulfamic acid: 60-100%), NALCO ® 79125 Safe Acid (sulfamic acid: 60-100%), and Rydlyme ® (hydrogen chloride: <10%), has been demonstrated to effectively control biofouling, being one of the most widespread treatments for cleaning and disinfecting Microorganisms 2023, 11, 1568 6 of 34 marine equipment and devices [43,55,56]. They can be applied through the immersion of equipment into disinfectant solutions or spray applications since these disinfectants are available in powder and/or tablet form. TermoRens ® Liquid 104 cleansing fluid (5-15% citric acid and <10% phosphoric acid) was formulated to remove mussels, barnacles, and additional marine organisms and is marketed as environmentally friendly. Likewise, Barnacle Buster ® (85% phosphoric acid) is promoted as a safe, non-toxic, and biodegradable marine growth removal agent [38]. In the peroxygen family, Virkon ® Aquatic is 99.9% biodegradable and breaks down to water and oxygen [57]. It is one of the very few U.S. Environmental Protection Agency registered disinfectants labeled specifically for use in aquaculture facilities against aquatic bacterial, fungal, and viral pathogens, and is available through aquaculture suppliers such as Syndel in North America [58,59]. In turn, in the European Community, Antec International Limited indicates that the compound is registered as a disinfectant only for professional use. Due to the restrictive legislation, which requires several risk studies before registration and marketing authorization, the global costs of the development of new biocides or new antifouling coatings incorporating biocides have increased [17]. These costs reactivated the development of non-toxic approaches, including novel antifouling surfaces in which some natural compounds can be incorporated. Although the choice of the correct strategy depends on the cost and application possibilities, antifouling coatings are probably the most cost-effective method for boats and other submerged devices and equipment [60,61].

Marine Coatings
Among all the strategies presented, novel modified surfaces and coatings probably represent the most cost-effective and promising methodology to tackle marine biofouling. These approaches include preventive measures for adhesion, biofilm formation, and development, and consequently delay macrofouler attachment and settlement. Since microfouling events can be managed directly by the performance of these surfaces/coatings, the effects of macrofoulers can be controlled more effectively. Antifouling coatings can be divided into chemically bioactive coatings and biocide-free coatings. The chemically active antifouling technologies, which act through the controlled release of bioactive molecules (most recently booster biocides), can be subdivided into three main categories: contact-leaching coatings, controlled-depletion paints (CDPs), and self-polishing copolymers (SPCs), and a few combinations thereof. All these technologies control the release of bioactive molecules via various chemical mechanisms, many of which remain partially understood [78]. From those, SPC coatings are the most successful antifouling coating technology in terms of longterm efficiency in service life, and where the biocidal compound is chemically bonded to the binder, which is gradually hydrolyzed and dissolved in water to release the antifouling bioactive agent. On the other hand, among the biocide-free coatings technologies, foulingrelease coatings (FRCs) are the most acceptable and implemented in the marine industry, mostly allied to their eco-friendly biocide-free antifouling effect, acting through mechanical and physicochemical mechanisms and providing long-term efficiency, particularly for dynamic systems (e.g., ships) [79].
Although the first SPC included TBT [47], novel coatings have been developed. In turn, with FRCs, biofouling may be removed by hydrodynamic stress through ship movement or mechanical cleaning. Although they prevent macrofouling events under dynamic conditions, FRCs are less effective in preventing the formation of the first adhesion layers [79].
In recent years, bioinspired antifouling strategies have emerged, including micro-and nanostructured surfaces, natural bioactive compounds, bioinspired hydrogels, slippery liquid-infused porous surfaces, bioinspired dynamic surfaces, and zwitterionic/amphoteric coatings [63,64]. Due to natural evolution, different organisms, including mussels, crabs, sharks, and insects, have demonstrated natural antifouling abilities in their bodies and structures [61]. Bioinspired coatings aim to mimic shapes, functions, and elements of nature. Since these promising antifouling coatings show practical value due to their environmental compatibility, they have been intensively explored to deal with marine biofouling. Biomimetic surfaces may be produced by several techniques, including deposition and electrostatic methods, 3D printing, self-assembly, and lithography, the most common methodology [63]. Most biomimetic coatings have been produced from soft polymers, such as polydimethylsiloxane (PDMS), poly(methyl methacrylate (PMMA), silicone, polyurethane, and polypropylene, since they present a low elastic modulus (a measure of a material's stiffness or resistance to elastic deformation under stress, calculated by the ratio of stress and strain, corresponding to the stress of the material) and low surface energy, allowing a fouling release effect [63]. Moreover, they are also inexpensive and chemically inert. Indeed, a surface based on shark skin comprising microscopic features (Sharklet AF TM ) was developed to prevent bacterial adhesion and biofilm development [80]. The drawbacks of biomimetic surfaces include the possibility of the designed nano-or microstructure being only active against specific fouling organisms, thus limiting the application range. Moreover, the antifouling effect may decrease after some time due to fouling organisms' attachment [61]. In addition, a low-cost and simple fabrication approach is required for marine applications [61].
Natural antifouling compounds obtained from invertebrates, plants, and microorganisms have also been proposed as one of the best alternatives to current chemical formulations in marine paints and coatings [66][67][68][69][70]81]. Antifouling mechanisms of these compounds may be related to alterations in protein expression (e.g., by promoting the underexpression of proteins related to adhesion and biofilm development), oxidative stress induction, neurotransmission blocking (caused by, for example, the inhibition of acetylcholine esterase activity, which interrupts cholinergic signaling and reduces the success of the settlement of fouling organisms), surface modification (e.g., by blocking the attachment site of bacteria), and biofilm inhibition through different mechanisms. However, the molecular mechanisms of action of these compounds are still under analysis. Compared to natural compounds obtained from higher organisms, such as crustacean shells and mollusks [82], those sourced from microorganisms present several benefits since they may be produced at a low cost by optimizing cultivation conditions [61,69]. Some of them are isolated from marine microorganisms [83], such as chitosan and melanin [84,85]. For example, antimicrobial peptides, commonly classified according to their source, charge, structure or residual pattern, and function (antibacterial, antibiofilm, antifungal, antiparasitic, insecticidal), include both membrane-acting and non-membrane-acting peptides [70]. The advantages of marine antimicrobial peptides include their stability in high salt concentrations and a range of temperatures (4 • C to 20 • C) [86]. Additionally, the use of extracts instead of purified compounds previously identified as active molecules could be a suitable approach due to lower production costs and the possibility of having different bioactive compounds in the same extract that may act synergically on different targets of fouler organisms [71,81].
Whales, fishes, and amphibians also secrete specific mucus that can prevent microbial adhesion, known as natural hydrogels [61,63]. Researchers have prepared synthetic hydrogels with a high degree of similarity to these natural hydrogels. Hydrogels are particularly hydrophilic 3D network structures of soft material that can absorb water, exhibiting a low interfacial free energy when in contact with liquid and a good resistance to protein adsorption [61,63]. Once a hydrogen bonding or an electrostatically induced hydration layer is formed on a hydrophilic surface, this constitutes a physical barrier to the adhesion and attachment of fouling organisms [87]. Synthetic hydrogels, such as polyethylene glycol (PEG), polyacrylamide (PAM), and polyurethane (PU), are usually fabricated by physical and chemical cross-linking methods [88]. However, improving the mechanical strength of hydrogels is required for their application in harsh marine environments. Filling hydrogels/polymers with nanomaterials and their modification with polymer brushes is an effective antifouling strategy since the brushes act as a steric barrier for bacteria and large molecules [14,63,89,90]. Recently, corals have been a subject of great interest for researchers as a novel source for exploring the potential of biomimetic surfaces [66]. Antifouling strategies from these organisms are related to the production of natural antifouling substances but are also due to their foul release, sloughing, and fluorescence effect. Indeed, the mucus produced by corals can protect them from biofouling by presenting a physical barrier, the production of antimicrobial compounds, and a slime sloughing effect. Furthermore, fluorescent corals emit a weak light that may prevent the attachment of diatoms. As no fluorescence effect was observed on bacteria, this strategy must be combined with additional ones to attain a broad-spectrum antifouling capability. However, the main drawbacks of corals are related to their natural environments. Since coral reefs are ecologically sensitive, their use may damage their ecosystems [66]. Although natural antifouling substances have been isolated from marine microbial organisms, invertebrates, algae, corals, and plants, chemical synthesis based on their composition is an alternative approach to tackle their limited production and extraction, which may hinder their large-scale production [61].
Slippery liquid-infused porous surfaces consist of a porous/textured material and lubricating liquid [63,64]. The advantages of these surfaces include the repellence of different liquids and resistance to ice and high pressures. These act as a physical barrier and a molecularly smooth surface, decreasing attachment strength and blocking signals with self-cleaning properties [63]. However, due to the complexity of the marine environment, the stability of these surfaces remains a challenge since the lubricant is easily lost under shear flow [64]. Dynamic surfaces, a changing surface that renews itself in seawater while removing fouling organisms, are an additional bioinspired strategy [63] using self-polishing and degradable copolymers. Finally, zwitterionic/amphoteric coatings are also a promising bioinspired approach [61,63,91]. The constituent of the lipid outer layer of the cell membrane, phosphatidylcholine, is an amphiphilic molecule comprising a hydrophilic head and a hydrophobic tail, showing great resistance to protein binding [61]. The phosphatidylcholine head groups are zwitterions consisting of equal numbers of oppositely charged species exhibiting neutral charge and a hydrophilic character. Zwitterionic polymers have the same number of cations and anions along their polymer chains [63], forming a strong hydration layer that impacts the initial deposition of proteins, contributing to their antifouling ability [14]. The main advantage of using zwitterionic polymer brushes in marine environments is that they are not affected by high concentrations of salt ions [63]. In turn, poor antifouling durability and mechanical strength are some of their limitations.
Polymer brushes are polymeric assemblies tethered at one end to a solid substrate either through covalent attachment or physical adsorption [92]. Antifouling polymer brushes have been developed to prevent the adsorption of molecules and adhesion by limiting the contact of the surface with the organism and reducing the force involved in bacterial attachment [93]. The immobilization of antimicrobial peptides, which present a broad spectrum of activity, on polymer brushes also represents a good approach to creating surfaces with antibacterial properties [70].
According to surface wettability, antifouling coatings may be considered hydrophilic, hydrophobic, or amphiphilic coatings. Hydrophilic coatings, such as hydrogels, form a hydrated layer that may bind water molecules so strongly that other molecules and fouling organisms cannot replace them during adhesion, thus preventing initial biofouling. However, their antifouling performance is not long-lasting, and their mechanical strength is usually low [64]. Hydrophobic coatings, such as PDMS, exhibit low surface energies, reducing the adhesion strength of fouling organisms on surfaces and allowing their easy removal [62]. However, due to the hydrophobic interaction between the slime compositions and hydrophobic surfaces, they cannot prevent the development of the first slime layer of the biofilm, which is mainly composed of proteins, bacteria, and diatoms [64]. Therefore, amphiphilic coatings, which are characterized by the presence of hydrophilic and hydrophobic groups, combine the advantages of hydrophilic and hydrophobic surfaces. Moreover, superhydrophobic surfaces, which present a high water contact angle, typically > 150 • , have also been tested as improvements for marine surfaces [94]. Studies performed by Ellinas et al. [95] and Kefallinou et al. [16] described hybrid metal-sputtered superhydrophobic surfaces demonstrating both bacterial repulsion and long-term killing efficacy against the cyanobacteria Synechococcus sp. PCC7942. Among the two low-surface-energy hydrophobic coatings used, a chlorosilane and a fluorocarbon coating, the fluorocarbon layer managed to better maintain the superhydrophobicity and anti-adhesiveness of the surface when enriched with an adequate amount of copper [16]. These bifunctional surfaces with antifouling and bactericidal activity can be a promising strategy for managing marine biofouling. Although several methods have been developed to produce superhydrophobic surfaces, including layer-by-layer assembly, electrodeposition, photolithography, electrospinning, and 3D printing, those based on simple chemical reactions are more attractive due to their simple procedure, low cost, and large-scale production potential [64].
Nanotechnology-based coatings, including the use of silver nanoparticles [96][97][98], carbon nanotubes (CNTs) [99][100][101][102], graphene [103][104][105][106][107], and metal oxide semiconductors such as titanium dioxide (TiO 2 ) [108] and zinc oxide (ZnO) [79], are relevant novel approaches to prevent biofouling. Photocatalytic antifouling coatings, based on the redox ability of semiconductor photocatalysts under light conditions such as TiO 2 and ZnO, showed good chemical stability and antifouling performance under ultraviolet conditions [62]. For example, ZnO can produce reactive oxygen species that induce oxidative stress and intracellular component outflow [64]. Marine coatings containing nanomaterials have been reported to be an efficient antifouling strategy offering hydrophobicity, water repellency, high durability, and anti-corrosive properties [65]. Moreover, nanocomposite coatings have good adhesion between the coating and the hull. Using a nanocomposite coating on the metal surface of a hull may eliminate the presence of holes, which can contribute to corrosion [64].
Although the main concern in marine coatings is related to their antifouling performance, the corrosion performance should also be considered [64]. Indeed, the adhesion and attachment of organisms on surfaces increase changes in the concentrations of ions, oxygen levels, redox potential, conductivity, and pH, which in turn prompt the biodegradation of coatings and stimulate chemical and electrochemical reactions between organisms, media, and metals. This type of corrosion, the microbially influenced corrosion, is responsible for about 20% of the corrosion occurring in aqueous environments. Amphiphilic polymers, bioinspired superhydrophobic surfaces, and slippery liquid-infused porous surfaces represent inherently integrated antifouling and anticorrosion coatings [64]. However, some modifications, such as the integration of polydopamine, graphene, polyaniline, and amorphous carbon, can be performed on other types of coatings to enhance their anticorrosion properties [64].
Due to the wide distribution of marine environments, a key question remains: how can the scientific community transfer in vitro knowledge from the laboratory to natural marine environments?

In Vitro Studies
Although numerous in vitro systems, including microtiter plates, the Calgary device, flow chambers and flow cells, the Robbins device, rotary biofilm reactors, and microfluidic devices, have been designed to study biofilm formation to better mimic real development conditions [12,13], few of them are characterized to be used in marine biofouling studies.
In vitro studies are very important since they are necessary as the first approach to evaluate the effectiveness of specific marine coatings on biofilms and to collect useful information for further in situ studies (Table 2). In vitro models can be operated in static or dynamic conditions. Although static models are simple and cheap and do not require specialized equipment, they often do not accurately represent many environmental conditions (e.g., the hydrodynamics) of natural marine environments [109]. In a dynamic model, nutrient supply and metabolite removal often occur throughout the process, resulting in longer-lasting operation. They also take into account the presence of hydrodynamic conditions, which have a real impact on biofilm development [110]. However, they often require specific equipment, higher costs, and technical competency due to their complexity of use [111].
In vitro models provide well-defined results, allowing precise control of experimental parameters while concomitantly allowing single variables to change. Consequently, this allows the study of the effects of single elements on various aspects of biofilm development. This simplistic approach is not possible for in situ models due to natural variations. However, they lack the interaction between different marine organisms since some species act synergistically through metabolite and signal production and/or direct contact, as well as all changes associated with environmental parameters that occur in real aquatic environments, such as pH, temperature, and hydrodynamic condition variations. Moreover, many in vitro studies are performed using non-representative artificial media [94,112] and hydrodynamic conditions [60,97]. This is particularly important since in marine environments, surfaces are in contact with a wide range of hydrodynamic conditions according to their location, and fouling organisms have evolved their ability to settle and proliferate on a range of surfaces, either under lower hydrodynamic conditions, such as a shear rate of 50 s −1 , reported for a ship in a harbor, and under high turbulent conditions, such as at 125,000 s −1 , the reported shear rate value for a navigating ship [113,114]. Likewise, hydrodynamic conditions also play a pivotal role in biofilm development since they affect biofilm architecture, diversity, EPS production, energy metabolism, and mass transfer, prompting molecular changes [30,[115][116][117]. While higher flow velocities improve molecular transport by convection, the higher density of biofilms decreases the diffusivity of the molecules inside them [118]. Furthermore, stronger shear forces are responsible for higher biofilm sloughing or detachment [119]. Indeed, to study how microalgae biofilms respond to different hydrodynamic conditions, the architecture and cohesion of Chlorella vulgaris biofilms were investigated in flow cells at three different shear stresses: 1, 6.5, and 11 mPa [2]. Biofilm cohesion was heterogeneous at low shear stress, resulting in a strong layer close to the substrate and more loose superficial ones. In turn, higher shear stress increased the cohesion of the biofilms allowing them to grow thicker and produce more biomass [2]. Using a microfluidic flow cell, the impact of shear stress on Cobetia marina and Pseudomonas aeruginosa biofilm formation was also evaluated [3]. The results indicated that hydrodynamics affect the biomass, maximum thickness, and surface area of biofilms, with the higher shear stress (5.6 Pa) promoting thinner biofilms than the lower shear stress (0.2 Pa). Particularly on cyanobacterial biofilms, studies performed on coccoid [120,121] and filamentous [122][123][124][125] cyanobacteria at controlled hydrodynamic conditions (values of shear rate of 4 s −1 and 40 s −1 ) showed a higher biofilm development at the lower shear rate. A study that aimed to evaluate the settlement of diatoms on different antifouling coatings also revealed that biofilm adhesion, diatom abundance, and diversity were found to be significantly different between static and dynamic treatments [126]. Therefore, due to the importance of hydrodynamic conditions on biofilm development, in vitro studies which aim to evaluate the performance of novel antifouling coatings [90,127] should mimic the typical real conditions that prevail in marine environments to bring the in vitro operational conditions closer to the natural aquatic environments [122]. Because of reactor geometry on the flow, the shear stress or shear rate should be considered to characterize shear effects/hydrodynamic conditions. The shear rate is the derivative of the velocity in the perpendicular direction from the wall system [128], quantifying the frequency at which cells contact the surface. The shear stress in Newtonian fluids is proportional to the shear rate, where the fluid viscosity is the constant of proportionality [128], representing the friction from the fluid acting on the adhered cells/biofilm. Computational fluid dynamics (CFD) is a commonly used approach to model biofilm reactors since it enables the faster estimation of the fluid flow parameters of these systems and at a relatively low cost in comparison to experimental techniques [129]. Since the results obtained from CFD comprise a larger number of points in the flow path, they provide much more detailed information about the flow field when compared with the experimental approach [113], although validation of the simulation results is required. Moreover, the standardization of biofilm reactors and operation conditions enables a rigorous comparison of hydrodynamic data obtained from different laboratories.
Microfluidic devices have demonstrated high potential and versatility for the study of biofilm formation under different growth conditions. These platforms allow the testing of different materials at highly controlled hydrodynamic conditions through a precise, non-invasive, and real-time analysis [130]. A microfluidic assay used to quantify how easily diatoms can be removed from several surfaces through shear force application showed that, while the number of adhered cells was barely affected by the different coatings, the critical shear stress required for their removal varied significantly [131]. Although these devices require small volumes to operate and can be custom-made for specific purposes, they require special equipment for manufacturing and operation. Moreover, clogging events can occur due to the small dimensions, air bubbles can have a very significant effect, and viscosity effects are also critical [13]. In addition to microfluidic devices [3,[131][132][133], platforms that specifically evaluate macrofouling development [134][135][136] have also been used in marine biofouling studies. Recently, a CFD analysis performed on agitated 12-well microtiter plates showed that this platform can be used as a marine biofilm reactor to mimic marine environments since the shear rate range achieved comprises the values found in real aquatic environments [122]. Indeed, the use of agitated microtiter plates at defined hydrodynamic conditions can be a very suitable and reasonable approach since this platform requires low volumes (and consequently has reduced costs) and is easy to handle. Furthermore, it enables the control of different parameters and the use of coupons from several materials to test the impact of different surfaces and coatings in marine biofouling, in a high-throughput mode (often required for studies performed during long time intervals with sampling on different days).
In vitro models still also face an important drawback related to the use of unrepresentative fouler organisms. Indeed, some of these studies, which aim to evaluate novel coatings to tackle marine biofouling, are performed using common model organisms for biofilm studies, such as Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Bacillus sp., and Candida albicans, but they are not considered relevant microfoulers in marine environments [4,107,137,138]. These models often use a single bacterial species, which is never the case in a natural environment since most biofilm communities are composed of multiple organisms living in proximity. Therefore, organism diversity should be considered in the evaluation of the performance of novel antifouling surfaces, as well as it is important to consider organisms with strong fouling activity and wide global distribution. However, some of the barriers to the in vitro study of mixed biofilms are related to the lack of knowledge about the abundance of each biofilm resident, which makes it difficult to select the correct initial concentration for in vitro assays, i.e., the difficulty in labeling different populations on biofilms (common issues are the stability of the tag and the influence it may have on the microorganism physiology) and the overall challenge in interpreting inter-species relations [1,139]. The relevance of using mixed populations instead of single cultures for in vitro screening assays of marine antifouling coatings was assessed in a recent study in which single-and dual-species biofilms of Pseudoalteromonas tunicata and a coccoid cyanobacterium were grown for 49 days on an epoxy resin [139], a marine coating with known antifouling potential [120]. The results obtained suggest that for initial screening, starting with a single representative organism such as a cyanobacterium is a good approach to predict the results obtainedin marine environments by in vitro testing. Indeed, while a marine bacterium alone revealed biofilm growth kinetics similar to dual-species biofilms, single-species biofilms presented a higher number of cells, biofilm wet weight, thickness, and biovolume when compared to dual-species biofilms [139]. Therefore, in that particular case, single-species cyanobacterial biofilms corresponded to the worst-case scenario for testing.
Although models that study early stages of biofouling formation are easier to implement due to the heterogeneity, complexity, and evolving nature of marine biofilms, the design of an accurate marine in vitro model is extremely challenging. To improve the evaluation of the performance of novel marine coatings, the characterization of marine reactors and the operational conditions that enable them to mimic, as closely as possible, the marine environment should be considered. Table 2. In vitro studies focused on different surfaces/coatings used in marine environments. The different surfaces/coatings were divided into non-modified surfaces, chemically bioactive coatings, biocide-free coatings, and a combined strategy of chemically bioactive coatings and biocide-free coatings. The distribution by rows follows a chronological order.

In Situ Studies
The marine environment is a complex habitat comprising up to 4000 potentially biofouling species [152]. Due to physiochemical intercommunication between different fouling species and all commensal, mutualistic, symbiotic, and additional relationships, in situ models can represent a more realistic approach than in vitro studies. Moreover, in situ marine biofilm studies allow the evaluation of biofilm properties under native conditions (undisrupted) and performing studies for a long time under natural conditions. Likewise, as commercial antifouling coatings should maintain antifouling capabilities for sometimes several years, in situ studies on natural marine environments may be particularly adequate [63]. Although there is no universal model for marine field tests, a minimum test period of six months is recommended since biofouling shows spatiotemporal variation under different seasons, temperatures, salinities, and light regimes [141], and limitations of the coatings will be revealed over a longer test period [64].
In turn, in situ studies usually require higher costs and specific equipment and devices related to the installation and sampling in natural marine environments. Moreover, sampling may be time-consuming and may be affected by natural conditions that, in some cases, are out of the control of the researchers, such as sea storms [153]. Most knowledge about biofouling and the performance of antifouling coatings has been conducted in the laboratory or in situ, in wave-protected habitats, usually in bays and port harbors. One of the main drawbacks related to in situ tests is the scarcity of studies performed under high-energy environmental conditions, such as under moderate and strong wave and current activity, due to logistical and safety-related difficulties in conducting detailed observations [153][154][155]. Since these high wave-energy regions of coastal oceans are becoming increasingly targeted as areas of human activity, such as aquaculture, and as a source of renewable energy, it is critical to improve knowledge about biofouling risks in these environments, as well as the evaluation of novel antifouling surfaces that can be used in the material design of relevant industrial equipment. Table 3 shows in situ studies focused on different marine surfaces/coatings developed for marine environments. Most in situ tests of novel marine coatings are performed after in vitro analyses to confirm if the effectiveness obtained under laboratory conditions is equivalent to what was achieved in natural marine environments [14,41,81,89,94,102,[106][107][108]137,145,151]. Although, in most cases, similar results are obtained between both tests [94,102,137,151], some contradictory findings have also been reported [14,89]. An in vitro study performed to test biomimicking micropatterned surfaces concluded that the settlement of barnacles on the patterned and smooth surfaces was similar [14]. However, in the field tests in natural seawater, barnacle settlement on the smooth surface was detected after 4 weeks of immersion, while no barnacles were observed on the patterned surfaces during the 7 weeks of the immersion period. Since it has been demonstrated that the antifouling properties of micropatterned surfaces may be associated with hydrodynamic forces, and the hydrodynamic conditions between the static laboratory and field tests were different, this may have contributed to the differences found [61,63]. Moreover, the discrepant period between in vitro (hours/days) and in situ (weeks/months) tests can also affect the performance of antifouling coatings [14,89]. Likewise, a study performed with pristine silicon rubber, graphene-added silicon rubber, and graphene-added silicon rubber filled with quaternary ammonium salt showed that the bactericidal graphene-added silicon rubber filled with quaternary ammonium salt coating exhibited an anti-adhesion effect under laboratory conditions, but the anti-adhesion effect was not durable since it lost antifouling effects completely in real marine conditions [106].
Unfortunately, few studies conduct a more realistic assessment of the performance of novel coatings due to the costs involved in the process [41]. After the determination of the minimal inhibitory concentrations (MICs) and minimal bactericidal concentrations (MBCs) of two commercial and functional biocides and the in vitro evaluation of biofilm adhesion potential of a marine bacterium on the surfaces with the immobilized biocides, in situ analyses were performed for up to 66 weeks in two different marine environments (Portugal and Singapore) [41]. Additionally, trial field tests on two coated ships with these formulations were accomplished. The ships traveled around the world (including Brazil, Cape Verde, and Greenland), experienced distinct ecosystems, and were also subjected to periodic dock stages. The analyses were performed after the ships had been traveling between eight and fourteen months and reflected the previous in situ results, corroborating biofilm adhesion performance, which demonstrates the predictive power of in situ testing [41]. Table 3. In situ studies focused on different surfaces/coatings used in marine environments in the last years. The different surfaces/coatings were divided into non-modified surfaces, chemically bioactive coatings, and a combined strategy of chemically bioactive coatings and biocide-free coatings. The distribution by rows follows a chronological order.      − Low and unique diversity was found in the copper-releasing coating − Differences were found between the two locations since the biofilm developed in Banyuls Bay was less dense compared to those formed in Toulon and presented a slower biofilm formation [168] Reduced graphene oxide/PDMS Graphene oxide-boehmite nanorod/PDMS composites Micro-and macrofoulers Tropical area 45 days, 23-28 • C − The higher self-cleaning and foul-release performance of the boehmite nanorod composite coating was observed [107] Abbreviations: CNTs-carbon nanotubes, Cu 2 O-copper oxide, CuPy-copper pyrithione, CuSCN-copper thiocyanate, FRCs-fouling-release coatings, MCE-methanol cell extract, MWCNTs-multi-walled carbon nanotubes, PDMAEMA-cationic polymer brush, PDMS-polydimethylsiloxane, PHEMA-co-PEG10MA-neutral polymer brush, PSBMA-zwitterionic polymer brush, PSPMA-anionic polymer brush, PVC-polyvinyl chloride, SWCNTs-single-walled carbon nanotubes, wt%-weight percent, ZnO-zinc oxide, ZnPy-zinc pyrithione. * Zinc oxide (ZnO) is not considered a biocide by regulations but was added in the formulation. ** Irgarol ® 1051 (N -tert-butyl-N-cyclopropyl-6-(methylthio)-1,3,5-triazine-2,4-diamine). *** Econea ® biocide (4-bromo-2-(4chlorophenyl)-5-(trifluoromethyl)-1H-pyrrole-3 carbonitrile).
Some in vitro models have evolved to mimic the real conditions in marine environments. However, in situ studies in real marine environments allow for long-time and surface tribological characterization, but they are also more complex. Overall, the main advantages and limitations of in vitro and in situ tests are described in Table 4.

In situ studies
Allow the study of complex interactions between marine organisms More expensive

Resemble natural marine conditions Sampling limitations by natural conditions
Allow the study of higher hydrodynamic conditions such as those found under high-energy environments.

Time-consuming sampling
Studies can be performed for a long time (months/years) and enable surface tribological characterization upon long immersion periods (friction coefficient, wear, temperature, durability of surfaces under harsh marine environments).
Requirement of specific equipment, devices, and specialized personnel related to the installation and sampling

Concluding Remarks
To date, there is no available universal strategy that is effective against marine biofouling. Compared to chemical treatment agents, fewer toxicological and environmental risks are often associated with non-chemical treatment agents. Successful solutions can be implemented from the combination of different strategies, such as the use of wipers with chemical compounds, which provide both physical and biological protection, or by the incorporation of UV radiation on a non-stick foul-release or self-polishing coating to match the performance of existing systems at reduced costs [45].
The improvement of environmentally friendly marine coatings such as protein-resistant polymers, FRCs, and bioinspired antifouling coatings is crucial for improved antifouling strategies. Advances in genetic tools may also provide a better understanding of the molecular mechanisms and biofilm-related functions [123][124][125], creating a high-throughput screening approach to find new targets for disrupting biofilms. In the progress of novel antifouling coatings, factors related to production, application, maintenance, and service life should be considered. Novel promising marine coatings should be non-toxic, effective in a wide range of applications, require low maintenance, have reduced cost, and maintain high performance over long periods [61]. Among chemically active antifouling technologies, SPC coatings are the most promising antifouling technology due to their long-term efficiency in service life. In turn, from the biocide-free coating approaches, FRCs are the most suitable for the marine industry due to their eco-friendly biocide-free attributes.
The heterogeneity and structural complexity of marine biofilms pose a great challenge to their evaluation and control. The initial in vitro screening of promising, novel coatings is an important step for selecting those that will be further tested in situ. Reliable in vitro models must strive to reproduce the environmental conditions present in marine systems, as these factors affect the biofilm structure, composition, and mechanical properties. While in vitro models are powerful tools for reproducibly testing the efficacy of different coatings and controlling some environmental parameters simultaneously, they fail to account for the complex and dynamic nature of the interactions that play out between marine organisms. Even though there is no gold-standard in vitro model for the study of marine biofouling, it is crucial to know the limitations of selected models so as to not over-extrapolate data and produce assumptions beyond the abilities of the model. A promising approach is to use in vitro testing using defined conditions that are relevant to the environmental scenario that is being mimicked (including the use of relevant organisms, media, hydrodynamics, etc.) as a screening tool and then proceed to in situ studies (by immersion) over extended periods to confirm the screening results. Further validation tests should include exposure to the actual working environment (for instance, using panels in a ship hull during its routine operation) to include the variability in shear conditions (docking and sailing periods) and the change in environmental conditions imposed by the geographical diversity found during the operation.
Overall, investing in the research and development of innovative technology that can provide practical and feasible tools to control biofouling while protecting the marine environment from harmful chemical and/or biological waste is essential. Therefore, economic factors and biosecurity risk-management decisions should be taken into consideration to contemplate the practicality, feasibility, and environmental impact of biofouling management options.