543 Natural Product Antifoulants and Coatings Development Dan Rittschof CONTENTS I. Overview 543 II. Fouling and Antifouling 544 A. The Scope of Fouling 544 B. Antifouling 546 C. Antifouling and the Environment 546 D. Experimental Approaches to Nontoxic Antifouling 547 III. Natural Products Antifoulants 550 A. Biological Targets and a Brief History of Natural Product Antifouling Studies 550 B. Mechanisms of Action of Natural Product Antifoulants 551 C. Toxic Mechanisms 554 D. Nontoxic Mechanisms 555 E. Quantitative Structure–Function Studies 555 F. Proof-of-Principle for Natural Product Antifoulants 555 IV. Ironies and Pitfalls 556 A. The Irony of Scholarly Work 556 B. Additional Technical Pitfalls 556 V. Work with the Commercial Sector 557 A. Technology Transfer 557 B. Commercialization 558 Acknowledgments 559 References 559 I. OVERVIEW The intent of this section is to provide an uncluttered overview and rapid reference to specific issues of interest. Every effort has been made to reference the rest of the text to enable entry into an extensive and detailed literature. My apologies to any and all researchers whose work has been unintentionally slighted. Fouling, the colonization of surfaces by abiotic and biotic substances and organisms, has molecular, microbial, and organismal levels of organization. All mechanisms of fouling involve molecular bonding interactions and molecules that act as inorganic and biological adhesives. Existing commercial technology intended to minimize or control impacts of fouling includes antifouling and foul-release approaches. Antifouling coatings technology is based upon mechanisms in which broad-spectrum biocides, usually toxic metal ions, kill organisms that settle on coatings. Many toxic coatings are designed 17 9064_ch17/fm Page 543 Tuesday, April 24, 2001 5:29 AM © 2001 by CRC Press LLC 544 Marine Chemical Ecology to slowly hydrolize so that surface erosion continuously presents toxic additives as well as polishes the surface to reduce drag forces. Foul-release coatings are usually based upon dimethyl silicone polymer technology. These coatings foul, but are designed to be cleaned easily. Foul-release coatings are usually catalyzed with organotin catalysts and are toxic until the catalyst leaches from the coating. These coatings often contain additives, such as oils and surfactants, that function as biocides. Antifouling coatings often have foul-release coating properties, and foul-release coatings have antifouling coating properties. Environmentally unacceptable consequences of the use of toxic organotins and heavy metals have prompted research on natural antifoulants. Natural antifoulants may result in impacts and issues of similar importance to the environment as those resulting from toxic metals. Laboratories worldwide now use bioassays with target fouling organisms to direct purification, identification, and development of antifoulant compounds. Most living organisms employ a variety of antifouling strategies, many of which are chemically based. Reports of natural compounds with antifoulant activity span over 40 years. Chemically, natural antifoulant compounds represent most classes of organic compounds. Although specific mechanisms of action are rarely reported, general mechanisms of action include toxins, anesthetics, surface-active agents, attachment and/or meta- morphosis inhibitors, and repellents. One concept addressed repeatedly by academic researchers is the use of compounds found in living organisms as active agents in antifouling coatings. Although natural antifoulants are common, development of functional coatings based upon natural products is a technological, financial, temporal, and regulatory nightmare. This is, in part, due to the biological impacts and chemical nature of the compounds. Other than broad-spectrum highly toxic compounds, individual additives are usually narrow spectrum in effectiveness. Prevention of fouling by one kind of organism is routinely supplanted by fouling of another type. Other challenges related to the development of viable, environmentally benign coatings that contain natural products relate to the chemical nature of compounds. These challenges include developing polymer systems compatible with the additives as well as with anticorrosive undercoats and appropriate mechanical and application properties. Development of commercial coatings using natural products is blocked by cost, the time horizon to meet government regulations, and meeting performance standards which are judged by compar- ison to coatings with unacceptable environmental impacts. For example, one new organic biocide was registered for use as an antifoulant in the United States in the last decade. In addition to taking 10 years, registration of this biocide cost millions of dollars. When one combines these time and monetary factors with the practice of holding new coatings to performance standards of coatings with environmentally unacceptable impacts, it is clear why there is little progress. Even if these political and economic constraints are addressed, it is unlikely, due to the diversity of fouling mechanisms, that nontoxic natural products will become the hoped-for broad-spectrum solutions. However, the potential is high for the development of environmentally benign solutions to fouling which combine natural products with degradable organic biocides. II. FOULING AND ANTIFOULING A. T HE S COPE OF F OULING Fouling encompasses processes that range from purely physicochemical and electrochemical to those of complex biology. At the molecular level, fouling consists of all standard bonding interac- tions and plating phenomena between molecules in solution and on a surface. 1,2,3 In natural waters, there are large numbers of biologically derived surfactants that bind to any surface. Surface-active molecules also partition at the air/water interface to form a fouling layer. 4 All surfaces submerged in the ocean are immediately subjected to molecular fouling. The physics of how moving water interacts with surfaces both explains why it is advantageous for organisms to attach to surfaces and provides insights into fouling, control limits, and antifouling 9064_ch17/fm Page 544 Tuesday, April 24, 2001 5:29 AM © 2001 by CRC Press LLC Antifoulants and Coatings 545 technology. Because water is viscous, considerable energy must be expended to move it. The combination of friction and high viscosity results in a layer (several centimeters to less than a millimeter) of water near a surface that is usually not moving and where diffusional processes dominate. This low energy environment provides a refuge from flow for weakly swimming plank- tonic organisms during the settlement phase. Planktonic organisms have adaptations that enable them to move out of water masses in which nutrients are depleted. Many plankton have the ability to migrate vertically and change water masses. 5,6 A large number of microorganisms (bacteria and diatoms) stop replicating and produce sticky exopolymers when nutrient levels drop in the water mass in which they are traveling. Propagules and micoorganisms are routinely small enough to fit in the boundary layer over most surfaces. 7 Even those with very poor bioadhesives are not exposed to forces that can dislodge them. This passive attachment has no behavioral component and occurs continuously in natural waters. 8 A final wrinkle in the mechanisms of macrofouling attachment is that molecular, micro-, and macrofouling propagules routinely aggregate in the water column in the absence of surfaces, forming long sticky strands that are carried to surfaces by flow and gravity. 9 If the new condition is high in nutrients, the phenotype of the microbe changes from sticky polymer/exopolymer production back to growth and replication. 10,11 Taking physics and energetics into account, attachment of living organisms to surfaces is advantageous because organisms can use environmentally generated water movement (energetically free to the organism) for feeding and waste removal. Delivery of toxic compounds from a surface is diffusionally driven and compromised by the decrease in toxin concentration proportional with the square of the distance from the surface. Even in thick boundary layers such as those found in very still conditions, convective flow further dilutes and carries toxins away. A consequence of these processes is that an organism attached to the surface, but that breathes and feeds in the water either above the boundary layer or at some distance from the diffusional source of toxin, is minimally exposed to toxins. This is part of the reason that organisms such as barnacles, with calcareous base plates that act as diffusion barriers, are such tenacious foulers and among the first to appear on a failing coating. Barnacles that survive the first few days on a surface grow sufficiently to feed and breathe in water with ineffective toxin levels. The spectrum of cues for attachment and attachment mechanisms that are used by macrofoulers is daunting, 8 as is the tenacity of many of their bioadhesives. 12–14 Attachment mechanisms can be characterized as a continuum representing successional fouling at one end of the spectrum and probability-driven fouling at the other extreme. In successional fouling, settlement of a macrofouling organism is dependent upon prior microfouling. Successional fouling has been clearly demonstrated for hydroids specialized to live on macroalgae and shells of hermit crabs, 15,16 as well as Hawaiian populations of the tube-building polychaete worm Hydroides elegans . 17 In contrast, with probability- driven fouling, organisms settle based upon the probability of their contact with surfaces and the probability of their settlement. Probability of contact depends upon the numbers of propagules available. Probability of settlement depends upon the physiological state of the organism and physical and physicochemical properties of the surface. Organisms such as barnacles, e.g., Balanus amphitrite , settle best on high surface energy unfouled surfaces. 18,19 In contrast to barnacles, there are organisms, such as abalone, 20 that require location of another species for settlement, and others, such as many hydroids ( Tubularia spp. and Eudendrum spp.), that settle passively on all surfaces. 21 Because fouled ships have visited various ports for years, many of the most common fouling organisms are now found in ports throughout the world. 22 Many of these cosmopolitan species — such as Enteromorpha spp., 23,24 calcareous tube worms such as Hydroides elegans , 17 bryozoans such as Bugula neritina , 25 hydroids such as Tubularia crocea , 21 and barnacles such as Balanus amphitrite 18 — are reproductive much of the year. These common foulers disperse as short-lived microscopic larvae, spores, or propagules that settle, metamorphose, and complete their life cycle in less than a month. Short propagule duration and rapid generation times may, in part, explain their successful colonization of the world’s harbors. These organisms are the weeds of the sea, 9064_ch17/fm Page 545 Tuesday, April 24, 2001 5:29 AM © 2001 by CRC Press LLC 546 Marine Chemical Ecology thriving out of their original ecological context. It is likely that new environmentally sound anti- fouling technology will be based upon an understanding of the physics, chemistry, and biology controlling colonization of surfaces by these organisms. A major factor controlling initial colonization is the nature of the surface. All surfaces have a physicochemical property called surface energy. 21 Surface energy is described by the way in which solvent molecules interact with a surface. It is surface energy, for example, that is responsible for water beading on some surfaces and spreading on others. Some surfaces, those with surface energies similar to dimethyl silane, have little ability to interact with biological adhesives and, thus, form poor adhesive bonds. 2 These surfaces are relatively easy to clean. The responses of fouling organ- isms to surface energy have been studied extensively, 4,7,9,21,26–31 and two generalizations can be made: (1) all surface energies foul, and (2) all possible patterns of colonization with respect to surface energy occur. Some organisms colonize surfaces with a range of surface energies. Some only colonize surfaces with a specific surface energy, and some colonize surfaces independent of surface energy. 21 Thus, if the local fauna is known, one can predict the kinds of fouling a surface with known surface energy will initially experience. However, the relationship gets rapidly muddled as time passes. Over time, surface energies on all submerged surfaces converge. 4,28 At the developing community level, other factors such as predation, competition between fouling organisms, and temporal larval availability further complicate the picture. 30 B. A NTIFOULING Fouling is combatted by a variety of cleaning techniques and by killing propagules. Killing is most effective either immediately before or immediately after propagules attach. By far, the most common antifouling techniques are based upon broad-spectrum biocides that kill settling organisms. Com- mon biocides include strong oxidants and metals such as copper, zinc, and tin. There are two basic mechanisms of toxic metal action. One is death by metal ion overload. Free metal ions are essential cofactors and usually in short supply. As a result, organisms have efficient active transport mech- anisms for obtaining metal ions, but mechanisms for shutting off their metal ion pumps have not evolved. In the presence of metal-based antifouling technology, organisms overload themselves with metals. The metals, in turn, disrupt their normal enzymatic and metabolic functions, and the organisms die. 32–34 The other mechanism is death by uncoupling of oxidative phosphorylation and electron transport. Organometal compounds such as tributyltin (TBT) are lipophylic. Lipophylic molecules partition into membranes and disrupt essential membrane functions, such as the electron transport processes required for generation of cellular energy through oxidative phosphorylation. 35 A fascinating sublethal effect of tributyltin is imposex, the development of male secondary and, in extreme cases, primary characteristics by females. Imposex leads to sterility in many molluscs or lack of reproduction because the population becomes all male. 36–40 C. A NTIFOULING AND THE E NVIRONMENT Toxic metals have long-term impacts on freshwater and marine environments. This is because metals are biologically conserved and recycled. Two major biological processes result in buildup of toxic metals in the environment: (1) continuous conservation by organisms of free ions such as copper and zinc, 32 and (2) “reorganification” of metals like tin. 35 Both processes result in conser- vation and buildup of toxic compounds until nontarget species are impacted. Although antifouling coatings containing TBT are very effective, 41 the negative impact of TBT and related metabolites on mariculture and wild-caught shellfish has resulted in regulations and bans on its use in countries that have established environmental policies. 42 The conclusion reached by regulators in most developed countries is that TBT released from antifouling coatings damages environmental health, impacting fisheries and aquaculture. Nonspecific effects are detrimental to quality of life and may threaten human health. The result is a pending worldwide ban to be in place 9064_ch17/fm Page 546 Tuesday, April 24, 2001 5:29 AM © 2001 by CRC Press LLC Antifoulants and Coatings 547 in 2003 and raging debate by members of the International Maritime Organization (IMO) on use of TBT antifouling coatings. Copper, though currently receiving less attention than organotin, will probably face similar restrictive legislation in the future. At this point, one developed country, Sweden, has banned all toxic metal antifouling coatings in its territorial waters, 43 and other countries such as the United States regulate application, removal, and waste disposal of toxic metal coatings. D. E XPERIMENTAL A PPROACHES TO N ONTOXIC A NTIFOULING Social and governmental responses to negative environmental impacts of toxic coatings have resulted in pressure to find alternatives. This pressure, combined with basic scientific curiosity about natural chemical control of fouling and the capability of laboratory rearing of fouling organisms for testing, led academic researchers to the study of natural antifouling mechanisms. One common hypothesis supported repeatedly is that many organisms continuously produce antifoulants, which is how they remain unfouled. 44–48 In some instances, the antifoulant produced is mucus. 49 In other instances, extractable organic compounds are known 50 or are novel secondary metabolites. 46,51 Initial searches for natural antifoulants involved biologists and natural products chemists. 52 Source organisms, such as tropical sponges and octocorals, were chosen because they do not foul when alive and because they are rich sources of novel secondary metabolites. 53–55 Similarly, organ- isms that were relatively easily cultured (diatoms, nudibranch larvae, and barnacles) were used for bioassays. Although the surfaces of living intact sponges and corals did not foul, molecules were extracted with organic solvents from the whole colony. 44,45 Only a few researchers 49,56–63 have concerned themselves with the activity found in the water surrounding, or on the surface, of intact organisms. Thus, there are really two broad classes of natural antifouling compounds: (1) com- pounds extracted from organisms that have antifouling activity which may never reach the surface of the organism, and (2) compounds found in the water-bathing organisms that have antifouling activity and are likely to serve a role in deterring growth of epibiota. 55 Many biological scholars consider that this second, more restrictive functional ecology approach is more likely to result in discovery of commercially viable compounds. 49,55,64 However, the relative merits of these approaches are unresolved, and the vast majority of compounds reported to possess antifouling activity are from organic extracts of whole colonies or organisms. Although the quest for natural antifouling compounds began about 50 years ago, citations in literature reviews are rare until the early and mid-1980s. 54,65 However, substantial funding for the natural product antifoulants research became available in the United States in the late 1970s and early 1980s. The Duke Marine Laboratory research team was the first to use mass-reared barnacles, Balanus amphitrite , in bioassays 45 and to direct isolation of antifoulant compounds. 18 Balanus amphitrite is a cosmopolitan hard-fouling organism introduced by shipping around the world. Barnacles and other hard foulers, such as oysters and tube worms, are logical targets because their tests are glued permanently to surfaces and, once attached, act as platforms for other fouling organisms. Since the late 1980s and early 1990s, funding for antifouling studies using natural products has been available around the world. Major contributions have been made, especially by Japanese and Australian research groups. The first comprehensive review of the topic is by Davis et al., 55 which compiles and synthesizes information from over 200 publications. Clare 46 adds substantial and comprehensive new information. Table 17.1 provides additional references from 1997 to the present. Since the development of bioassays, there has been little change in the approach to discovering natural product antifoulants. The first step is showing that an organic extract of some kind of organism prevents settlement of a target species. 66 Then, depending upon the potency, stability, and maturity of chemical techniques for determining that particular class of natural product and the availability of the techniques to the investigators, there are varying levels of success in determining the structure of the active molecule. Initially, steroids, 67 terpenes, 44,51 phenolics, 68 bromonated 9064_ch17/fm Page 547 Tuesday, April 24, 2001 5:29 AM © 2001 by CRC Press LLC 548 Marine Chemical Ecology TABLE 17.1 Some Reports of Natural Product Antifoulants from 1995–Present Extract or Parent Compound (Number of New Structures) Source Bioassay Potency (EC 50 ) Reference Diterpene (3) Sponge Barnacle Settlement 0.45-1.1 µ g/ml 147 Ca(OH) 2 Concrete Oysters — 148 Nitrogen Hetrocycle Sponge Barnacle Settlement Bryozoan Settlement Byssal Thread Attach. 25 µ g/cm 2 1.1 µ g/cm 2 1.2 4.4 µ g/cm 2 121 Terpenoids (32) Sponge Nudibranch Barnacle Settlement 0.08–>50 µ g/ml 142 Terpenoids (14) Sponge Barnacle Settlement <0.5–0.05 µ g/ml 149 Film (3) Bacteria Barnacle Settlement Bryozoan — — 75 Diterpene (9) Sponge Barnacle Settlement 0.08–4.6 µ g/ml 150 Sesquiterpene (3) Nudibranch Barnacle Settlement 0.13–0.14 µ g/ml 150 Spermidine (10) Sponge Barnacle Settlement 0.1–15 µ g/ml 151 Cyanoformamide (2) Sponge Barnacle Settlement Cytotoxicity 4.3–5 µ g/ml 2.1–3.4 µ g/ml 152 BromoTyrosine (8) Sponge Barnacle Settlement (7) Ascidian Met. Promote (2) Cytoxicity (2) 0.1–8.0 µ g/ml 1.2–25 µ g/ml 2.1–3.4 µ g/ml 77 Oroidin (2) (1) Sponge Barnacle Settlement Ascidian (Promoter) 15–19 µ g/ml 2.5 µ g/ml 153 Steroid (2) Sponge Antimicrobial 10 µ g/disc 154,155 Aqueous Extract Film Bacteria Bryozoan Settlement — 97 Phlortannins (3) Marine Algae Polychaete Worms Bacteria 0.5–5 µ g/ml — 156 Aqueous Extract (Homarine) Soft Coral Antimicrobial 1.26 µ g/ml 60 Polyacetylene (7) Sponge Barnacle Settlement 0.24–4.5 µ g/ml 78 Terpenes (6) and Steroids (2) Sponge Barnacle Settlement 0.24–4.0 µ g/ml 154 Bromophenol (3) Polychaete Bivalves and Polchaete Settlement Field 5–240 ng/g 59 Lentil Lectin Plants Barnacle Settlement Inhibition 50–100 µ g/ml 157 Protein Complex (SIPC) Barnacle Larval Settlement Inducer 10–100 µ g/ml 106 Spermidine (10) Sponge/Synthetic Antimicrobial 8–256 µ g/ml 115 Diterpene (4) Algae Bryozoans, Hydroid >1 µ g/ml 61 Steroid (4) Sponge Anti-barnacle Settlement 5 µ g/ml 113 Terpenoids (14) Sponge Barnacle Settlement 50 mg/ml 113 Cyanoformamide (2) Sponge Barnacle Settlement 5 µ g/ml 158 Calmodulin Inhibitors Synthetic Barnacle Settlement Inhibition 1–10 µ M 159 Neurotransmitter Blockers (3) Synthetic Barnacle Settlement 30–300 µ g/ml 111 Terpene Analogues (19) Synthetic Barnacle Settlement 1 ng–5 µ g/ml 84 Neuroactive Compounds Synthetic Mussel Metamorphosis Induction 100 µ M 160 Steroid (4) Octocoral Anti-barnacle Settlement 2 µ g/ml 161 Steroid (4) Octocoral Lethal to Barnacle Larvae 100 µ g/ml 162 Lumichrome Ascidian Ascidian Metamorphosis Inducer 100 nM 112 9064_ch17/fm Page 548 Tuesday, April 24, 2001 5:29 AM © 2001 by CRC Press LLC Antifoulants and Coatings 549 hydrocarbons, 20,59 brominated tyrosine derivatives, 69 and saponins 70 were reported to act as antifou- lants at some level. Since the early 1990s, there has been a dramatic increase in research looking for potential antifoulants. 46 There are, or were until very recently, very active research groups in Australia (Kjelleberg, Steinberg, and associates), the Netherlands (TNO), Japan (Fusetani Biofouling Project), and the United Kingdom (Callow, Clare, and others). Additionally, there are smaller efforts in the United States, Singapore, India, New Caledonia, and Hong Kong. Major productive efforts, espe- cially by the Fusetani research group in Japan and the European and Australian research commu- nities employing bioassays-directed purifications, have resulted in identification of many new antifoulant compounds from a variety of marine invertebrates. In a review, Clare 46 reported over 50 core natural product structures with antifouling activity. Since 1995, the research community has reported the identity of over 100 additional natural products with antifouling activity (Table 17.1). Reports of many more compounds are delayed because of issues related to the process of patent protection, required for any subsequent commercialization. Inspection of Table 17.1 is useful in understanding the strengths and weaknesses of what has become a worldwide quest. Of the more than one hundred compounds shown in Table 17.1, the vast majority, about four-fifths, are terpenoid compounds, their relatives, or analogues. Most of these compounds are new to science since 1995. In over half of the studies, the bioassay used is for prevention of settlement of an easily culturable barnacle, 71 a major fouling species common to temperate and tropical harbors around the world, Balanus amphitrite. 72 Although B. amphitrite is a dominant fouling barnacle and an excellent test organism, it is known that it cannot be used as a representative of all fouling organisms or even, for that matter, of all balanoid barnacles. 73–75 Several authors have observed that potent antibarnacle settlement has no effect on other fouling species 76 or actually stimulates settlement. 77,78 From the perspective of potency for theoretical use in an antifouling coating, the vast majority of compounds in Table 17.1 exceed the potency criterion for future study that was followed in the U.S. Navy program, that of being active at less than 25 µg/mL in static bioassays. However, the notion of harvesting organisms and purifying commercial amounts of natural products for virtually any commercial use is probably untenable 79 and extremely remote for natural product antifou- lants. 46,80–82 One potential solution is the use of synthetic analogues developed from structure- function studies. 44,80–84 The idea here is to find commercially practical alternatives. In general, it is unlikely that natural products will be in sufficient quantity that they can be harvested, especially from the natural sources that are often exotic rare corals, sponges, and other invertebrates. Most potent natural product compounds are often too structurally complex to be commercially synthe- sized. Alternative compounds must have a potency that makes them practical and must be amenable to cost effective synthesis. Neurotransmitter Blockers (6) Synthetic Barnacle Settlement 0.1–100 µM 163 Homoserine Lactone Algae Bacterial Movement — 127 Aqueous Extracts Synthetic Barnacle Settlement — 19 Cardenolide (5) Insects/Plants Barnacle Settlement 10–50,000 µg/ml 48 Biogenic Amine Synthetic Bryozoan Metamorphosis Inhibitor 100 µM 164 TABLE 17.1 (CONTINUED) Some Reports of Natural Product Antifoulants from 1995–Present Extract or Parent Compound (Number of New Structures) Source Bioassay Potency (EC 50 ) Reference 9064_ch17/fm Page 549 Tuesday, April 24, 2001 5:29 AM © 2001 by CRC Press LLC 550 Marine Chemical Ecology III. NATURAL PRODUCTS ANTIFOULANTS A. B IOLOGICAL TARGETS AND A BRIEF HISTORY OF NATURAL PRODUCT A NTIFOULING STUDIES As the natural products antifouling field matures, the nature and diversity of organisms used in fouling assays is increasing, as is our understanding of the complexity of the biological processes involved. Initially, researchers assumed that fouling was a successional process and that microbial fouling was a requirement for macrofouling. This concept originated mainly from two sources: (1) a Meadows and Williams paper in Nature, 85 and (2) a paper by Corpe. 86 The Nature paper 85 specifically addressed settlement by polychaetes, and the assumption of successional fouling appears correct for many polychaetes. 17,85,87,88 Corpe 86 never suggested that microfouling was req- uisite for macrofouling, but his work was interpreted that way. Thus, initially it was postulated, that if microbial fouling could be controlled, then macrofouling would also be controlled. A statement to this effect can be found in virtually every report on control of microfouling far into 89 stimulate settlement of bryozoans on some surfaces 90 and settlement of bivalves like oysters 91,92 on others, and that bacterial films may have a variety of effects, ranging from nothing to inhibiting barnacle settlement. 19,75,93–97 However, temporal aspects on the level of weeks may result in changes in the effects of films on settlement. Perhaps the best way to view this phenomenon is that bacterial films have a major impact on most fouling organisms, 98,99 but preventing microfouling will not prevent macrofouling. Other chapters in this volume treat this topic in detail. Many macrofoulers readily settle on surfaces whether or not they are filmed with bacteria, and chemical surface characteristics mediate the settlement of many common macrofoulers. 9,21,28,29 Finally, at all levels of fouling, there are some organisms that settle like dust on all surfaces. 8,21,55 The major early exception to using microfoulers instead of macrofouling organisms in fouling studies was the use of settlement stage barnacles. Barnacles were targeted initially because they were recognized as central to the failing of antifouling coatings. 71,72,100 Barnacles are dominant members of fouling communities around the world and are easily recognized during visual inspec- tion and cleaning of fouled hulls and surfaces. Barnacles are calcareous (hard foulers) and have a relatively robust adhesive. Since cyprid glue contains barnacle settlement pheromones, 101,102 even if the first wave of larvae dies, the glue and tests left behind stimulate settlement of other barnacle larvae while at the same time acting as a barrier to toxin diffusion. If the relatively vulnerable cyprid survives long enough to settle and metamorphose, the newly recruited “pin head” barnacle has its own larval glue and uncalcified base plate to act as barriers to diffusion from below. The top of the pin head barnacle is elevated approximately 500 µm over the surface, just high enough to enable the barnacle to ventilate in water with reduced levels of toxins. 7,103 The base plate and a growth form, which results in breathing and feeding in diffusionally limited toxicity associated with the boundary layer of the hull surface, often results in barnacles being among the first to settle and survive on toxic surfaces. In addition to cyprids, 101,104 settled barnacles also produce settlement pheromones 102,105,106 thereby furthering recruitment. The result is that barnacles are often the first macrofoulers to colonize a protected surface. 100 Once growth of barnacles on toxic metal coatings is initiated, the surface rapidly fails because barnacles act as nontoxic platforms for other organisms. As a result of these kinds of field evidence, the U.S. Navy supported basic barnacle research for almost four decades, and barnacles were the only macrofoulers included in initial studies of natural product antifoulants supported by the U.S. Navy. Most of this work was conducted by the Costlow group at the Duke University Marine Laboratory. 47 At Duke in the early 1980s, mass cultured barnacle larvae were used as the target organism in bioassay directed studies of natural inhibitors of fouling. Approximately 70% of the marine organisms 9064_ch17/fm Page 550 Tuesday, April 24, 2001 5:29 AM © 2001 by CRC Press LLC ). There is also ample evidence that bacterial films can the 1990s (see for example, Readman Antifoulants and Coatings 551 in the temperate to subtropical estuarine communities around the Marine Laboratory were found to contain compounds with readily detectable antibarnacle settlement activity. Many compounds are extremely potent in the settlement assay, even though they have low toxicity. 94,107,108 The prevalence of compounds that interfere with settlement and metamorphosis of complex organisms like barnacles is a logical consequence of the complexity of the biochemical pathways controlling metamorphosis. There is evidence that these pathways begin with chemoreceptors cou- pled to neuronal, hormonal, and metabolic control processes through classic amplification cascades that include second messengers 20,22,88,102 that are stimulated by changes in ion permeability. 109 B. MECHANISM OF ACTION OF NATURAL PRODUCT ANTIFOULANTS The mechanisms of action of most compounds is not obvious from their structures. Although certain major classes of molecules, such as steroids, are recognized to have known functions, molecules are usually multifunctional. Examination of the structures presented illustrate this point. Compound 17.1 is a sodium/potassium ATPase inhibitor and a potent natural product toxin. 110 Compound 17.2 is a component of the fragrance of peaches and apricots and somehow anesthetizes barnacle larvae. 84 Compound 17.3 is reported to be a repellant of polychaete larvae. Compounds 17.4–17.7 have unreported mechanisms of action. One might recognize that Compound 17.4 has many of the characteristics of sesquiterpene antifoulants, which act via an unknown mechanism, and hazard that Compounds 17.5–17.9 might function through interaction with a surface or act like detergents. OH OH HO Calitriol C 0.24 µg/mL (17.5) Br Br (CH 2 ) 2 NHCHO H N O HO N O Br CH 3 O Br O O Ceratinamide A 100 ng/mL (17.4) Br Br Br OH Tribromophenol 3 ng/g (17.3) O O Pentyl 2-Furyl Ketone 1ng/mL (17.2) HO CH 3 CH 3 O H OH O Bufalin 10pg/mL (17.1) 9064_ch17/fm Page 551 Tuesday, April 24, 2001 5:29 AM © 2001 by CRC Press LLC 552 Marine Chemical Ecology As molecules increase in complexity, the number of possibilities for mechanism of action also increase. For example, Structure 17.10 inhibits a specific kinase and specific cellular second messenger. 111 In contrast, Structure 17.11 is specifically related to vitamin B2, 112 while Structure 17.12, a steroid peroxide, 113 is, in the imagination of this author, a molecule likely to impact steroid receptors and enzymes like cytochrome p450s involved in steroid metabolism. However, it is likely that the multiple functions observed for many complex natural products are a result of their interaction with multiple pathways and mechanisms. The environmental fates and NH 2 H N NH 2 Spermadine-antimicrobial (17.9) O S Br Br OH OH Br Br O O Bromophenol (17.8) NaO 3 SO NaO 3 SO OSO 3 Na Halistanol Sulfate 10 µg/disk (17.7) NHCHO H H O Cl Kalihinene X 0.5 µg/mL (17.6) N N O O H Vinocetine (17.10) N N NH H N O O Lumichrome (17.11) HO O O Sterol Peroxide (17.12) 9064_ch17/fm Page 552 Tuesday, April 24, 2001 5:29 AM © 2001 by CRC Press LLC [...]... long-term studies, and fates and effects of bioactive additives A few examples resulting from similar approaches to biocides include dichloro-diphenyl-trichloroethane (DDT), mercury, lead arsenic, cadmium, and TBT A OH NH2 NH2 HO HO OH OH DOPA (17. 13) Norepinephrine (17. 14) O N HN H N N H HO HN NH2 Serotonin (17. 15) Lisuride (17. 16) OH H N HO OH Isoproteranol (17. 17) © 2001 by CRC Press LLC 9064_ch17/fm... LLC 9064_ch17/fm Page 566 Tuesday, April 24, 2001 5:29 AM 566 Marine Chemical Ecology 150 Okino, T., Yoshimura, E., Hirota, H., and Fusetani, N., New antifouling kalihipyrans from the marine sponge Acanthella cavernosa, J Nat Prod., 59, 1081, 1996 151 Tsukamoto, S., Kato, H., Hirota, H., and Fusetani, N., Pseudoceratidine: a new antifouling spermidine derivative from the marine sponge Pseudoceratina... R., Eds., Oxford & IBH Publishing, New Delhi, India, 1988, 609 © 2001 by CRC Press LLC 9064_ch17/fm Page 562 Tuesday, April 24, 2001 5:29 AM 562 Marine Chemical Ecology 58 Woodin, S.A., Marinelli, R.L., and Lincoln, D.E., Allelochemical inhibition of recruitment in a sedimentary assemblage, J Chem Ecol., 19, 517, 1993 59 Woodin, S.A., Lindsay, S.M., and Lincoln, D.E., Biogenic bromophenols as negative... Targett, N.M., and Schulte, B., Chemical ecology of marine organisms: an overview, J Chem Ecol., 12, 951, 1986 55 Davis, A.R., Targett, N.M., McConnell, O.J., and Young, C.M., Epibiosis of marine algae and benthic invertebrates: natural products chemistry and the mechanisms inhibiting settlement and overgrowth, in Marine Bioorganic Chemistry, Vol 3., Scheuer, P.J., Ed., Springer-Verlag, Berlin, 1989, 85... potential F PROOF-OF-PRINCIPLE FOR NATURAL PRODUCT ANTIFOULANTS At present, compounds with promise must move through a torturous and daunting process if they are to be commercialized The process begins with proof-of-principle testing for patenting A standard predicament is that the researcher with the natural product is not familiar with polymer coatings and associated technology Thus, proof-of-principle... © 2001 by CRC Press LLC 9064_ch17/fm Page 564 Tuesday, April 24, 2001 5:29 AM 564 Marine Chemical Ecology 105 Rittschof, D., Oyster drills and the frontiers of chemical ecology: unsettling ideas, Am Malac Bull., special ed., 1, 111, 1985 106 Matsumara, K., Nagano, M., and Fusetani, N., Purification of a larval settlement-inducing protein complex (SIPC) of the barnacle, Balanus amphitrite, J Exp Zool.,... © 2001 by CRC Press LLC 9064_ch17/fm Page 560 Tuesday, April 24, 2001 5:29 AM 560 Marine Chemical Ecology 12 Waite, J.H., Adhesion in bysally attached bivalves, Biol Rev., 58, 209, 1983 13 Waite, J.H., Jensen, R.A., and Morse, D.E., Cement precursor proteins of the reef-building polychaete Phragmatopoma californica (Fewkes), Biochem., 31, 5733, 1992 14 Yamamoto, H., Marine adhesive proteins and some... researcher with the natural product is not familiar with polymer coatings and associated technology Thus, proof-of-principle is generally done in short-term studies © 2001 by CRC Press LLC 9064_ch17/fm Page 556 Tuesday, April 24, 2001 5:29 AM 556 Marine Chemical Ecology using either no polymer matrix at all128 or matrices like abscisic acid,129 phytogel130,131 or, in the more sophisticated cases, derivatives... J.C., Bowden, B.J., Tapiolas, D.M., and Dunlap, W.C., In situ isolation of allelochemicals released from soft corals (Coelenterata: Octocorallia): a totally submersible sampling apparatus, J Exp Mar Biol Ecol., 17, 69, 1982 57 Targett, N.M., Allelochemistry in marine organisms: chemical fouling and antifouling strategies, in Marine Biodeterioration, Thompson, M., Sarojini, R., and Nagabhushanam, R., Eds.,... for this and related topics can be found in a new special issue of Biofouling, vol 15, 2000, that is a © 2001 by CRC Press LLC 9064_ch17/fm Page 558 Tuesday, April 24, 2001 5:29 AM 558 Marine Chemical Ecology compilation of reports from the 10th International Congress on Marine Corrosion and Fouling in Melbourne, Australia, 1999.) It is clear from the Melbourne meeting that there is an elevated level . A H N OH HO Isoproteranol (17. 17) OH N N HN O HN H Lisuride (17. 16) HO Serotonin (17. 15) H N NH 2 NH 2 OH HO Norepinephrine (17. 14) OH NH 2 OH HO DOPA (17. 13) 9064_ch17/fm Page 553 Tuesday, April. (17. 5) Br Br (CH 2 ) 2 NHCHO H N O HO N O Br CH 3 O Br O O Ceratinamide A 100 ng/mL (17. 4) Br Br Br OH Tribromophenol 3 ng/g (17. 3) O O Pentyl 2-Furyl Ketone 1ng/mL (17. 2) HO CH 3 CH 3 O H OH O Bufalin 10pg/mL (17. 1) 9064_ch17/fm Page 551 Tuesday, April. 1988, 609. 9064_ch17/fm Page 561 Tuesday, April 24, 2001 5:29 AM © 2001 by CRC Press LLC 562 Marine Chemical Ecology 58. Woodin, S.A., Marinelli, R.L., and Lincoln, D.E., Allelochemical inhibition