Marine Chemical Ecology - Chapter 17 doc

Antifouling coatings technology is based upon mechanisms in which broad-spectrum biocides, usually toxic metal ions, kill organisms that settle on coatings... Successional fouling has be

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

544 Marine Chemical Ecology

to slowly hydrolize so that surface erosion continuously presents toxic additives as well as polishesthe surface to reduce drag forces Foul-release coatings are usually based upon dimethyl siliconepolymer technology These coatings foul, but are designed to be cleaned easily Foul-release coatingsare usually catalyzed with organotin catalysts and are toxic until the catalyst leaches from thecoating These coatings often contain additives, such as oils and surfactants, that function asbiocides Antifouling coatings often have foul-release coating properties, and foul-release coatingshave antifouling coating properties Environmentally unacceptable consequences of the use of toxicorganotins and heavy metals have prompted research on natural antifoulants Natural antifoulantsmay result in impacts and issues of similar importance to the environment as those resulting fromtoxic 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 withantifoulant activity span over 40 years Chemically, natural antifoulant compounds represent mostclasses of organic compounds Although specific mechanisms of action are rarely reported, generalmechanisms 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 inliving 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 chemicalnature of the compounds Other than broad-spectrum highly toxic compounds, individual additivesare usually narrow spectrum in effectiveness Prevention of fouling by one kind of organism isroutinely supplanted by fouling of another type Other challenges related to the development ofviable, 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 ison to coatings with unacceptable environmental impacts For example, one new organic biocidewas registered for use as an antifoulant in the United States in the last decade In addition to taking

compar-10 years, registration of this biocide cost millions of dollars When one combines these time andmonetary factors with the practice ofholding new coatings to performance standards of coatingswith environmentally unacceptable impacts, it is clear why there is little progress Even if thesepolitical and economic constraints are addressed, it is unlikely, due to the diversity of foulingmechanisms, 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 foulingwhich 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 tothose 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 Innatural waters,there are large numbers of biologically derived surfactants that bind to any surface Surface-activemolecules 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 advantageousfor organisms to attach to surfaces and provides insights into fouling, control limits, and antifouling9064_ch17/fm Page 544 Tuesday, April 24, 2001 5:29 AM

Antifoulants and Coatings 545

technology Because water is viscous, considerable energy must be expended to move it Thecombination of friction and high viscosity results in a layer (several centimeters to less than amillimeter) of water near a surface that is usually not moving and where diffusional processesdominate 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 whichnutrients are depleted Many plankton have the ability to migrate vertically and change watermasses.5,6 A large number of microorganisms (bacteria and diatoms) stop replicating and producesticky 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 mostsurfaces.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-, andmacrofouling 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/exopolymerproduction back to growth and replication.10,11

Taking physics and energetics into account, attachment of living organisms to surfaces isadvantageous because organisms can use environmentally generated water movement (energeticallyfree 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 withthe square of the distance from the surface Even in thick boundary layers such as those found invery still conditions, convective flow further dilutes and carries toxins away A consequence ofthese processes is that an organism attached to the surface, but that breathes and feeds in the watereither above the boundary layer or at some distance from the diffusional source of toxin, is minimallyexposed to toxins This is part of the reason that organisms such as barnacles, with calcareous baseplates that act as diffusion barriers, are such tenacious foulers and among the first to appear on afailing coating Barnacles that survive the first few days on a surface grow sufficiently to feed andbreathe 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 becharacterized as a continuum representing successional fouling at one end of the spectrum andprobability-driven fouling at the other extreme In successional fouling, settlement of a macrofoulingorganism is dependent upon prior microfouling Successional fouling has been clearly demonstratedfor hydroids specialized to live on macroalgae and shells of hermit crabs,15,16 as well as Hawaiianpopulations 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 theprobability of their settlement Probability of contact depends upon the numbers of propagulesavailable Probability of settlement depends upon the physiological state of the organism andphysical 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, thereare 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.21Because fouled ships have visited various ports for years, many of the most common foulingorganisms 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 bryozoanssuch as Bugula neritina,25 hydroids such as Tubularia crocea,21 and barnacles such as Balanus

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, explaintheir 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

546 Marine Chemical Ecology

thriving out of their original ecological context It is likely that new environmentally sound fouling technology will be based upon an understanding of the physics, chemistry, and biologycontrolling colonization of surfaces by these organisms

anti-A major factor controlling initial colonization is the nature of the surface anti-All surfaces have aphysicochemical property called surface energy.21 Surface energy is described by the way in whichsolvent molecules interact with a surface It is surface energy, for example, that is responsible forwater beading on some surfaces and spreading on others Some surfaces, those with surface energiessimilar to dimethyl silane, have little ability to interact with biological adhesives and, thus, formpoor 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 bemade: (1) all surface energies foul, and (2) all possible patterns of colonization with respect tosurface energy occur Some organisms colonize surfaces with a range of surface energies Someonly colonize surfaces with a specific surface energy, and some colonize surfaces independent ofsurface energy.21 Thus, if the local fauna is known, one can predict the kinds of fouling a surfacewith 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 developingcommunity level, other factors such as predation, competition between fouling organisms, andtemporal 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 mosteffective either immediately before or immediately after propagules attach By far, the most commonantifouling 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 basicmechanisms of toxic metal action One is death by metal ion overload Free metal ions are essentialcofactors 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 notevolved In the presence of metal-based antifouling technology, organisms overload themselveswith metals The metals, in turn, disrupt their normal enzymatic and metabolic functions, and theorganisms die.32–34 The other mechanism is death by uncoupling of oxidative phosphorylation andelectron transport Organometal compounds such as tributyltin (TBT) are lipophylic Lipophylicmolecules partition into membranes and disrupt essential membrane functions, such as the electrontransport 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, inextreme cases, primary characteristics by females Imposex leads to sterility in many molluscs orlack 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 becausemetals 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 ascopper 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 TBTand related metabolites on mariculture and wild-caught shellfish has resulted in regulations andbans 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 damagesenvironmental health, impacting fisheries and aquaculture Nonspecific effects are detrimental toquality of life and may threaten human health The result is a pending worldwide ban to be in place9064_ch17/fm Page 546 Tuesday, April 24, 2001 5:29 AM

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 similarrestrictive legislation in the future At this point, one developed country, Sweden, has banned alltoxic metal antifouling coatings in its territorial waters,43 and other countries such as the UnitedStates 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 naturalchemical 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 hypothesissupported repeatedly is that many organisms continuously produce antifoulants, which is how theyremain unfouled.44–48 In some instances, the antifoulant produced is mucus.49 In other instances,extractable organic compounds are known50 or are novel secondary metabolites.46,51

Initial searches for natural antifoulants involved biologists and natural products chemists.52Source organisms, such as tropical sponges and octocorals, were chosen because they do not foulwhen 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 forbioassays Although the surfaces of living intact sponges and corals did not foul, molecules wereextracted with organic solvents from the whole colony.44,45 Only a few researchers49,56–63 haveconcerned themselves with the activity found in the water surrounding, or on the surface, of intactorganisms 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 antifoulingactivity and are likely to serve a role in deterring growth of epibiota.55 Many biological scholarsconsider that this second, more restrictive functional ecology approach is more likely to result indiscovery of commercially viable compounds.49,55,64 However, the relative merits of theseapproaches are unresolved, and the vast majority of compounds reported to possess antifoulingactivity are from organic extracts of whole colonies or organisms

Although the quest for natural antifouling compounds began about 50 years ago, citations inliterature reviews are rare until the early and mid-1980s.54,65 However, substantial funding forthe natural product antifoulants research became available in the United States in the late 1970sand early 1980s The Duke Marine Laboratory research team was the first to use mass-rearedbarnacles, Balanus amphitrite, in bioassays45 and to direct isolation of antifoulant compounds.18

world Barnacles and other hard foulers, such as oysters and tube worms, are logical targetsbecause their tests are glued permanently to surfaces and, once attached, act as platforms forother fouling organisms

Since the late 1980s and early 1990s, funding for antifouling studies using natural products hasbeen available around the world Major contributions have been made, especially by Japanese andAustralian research groups The first comprehensive review of the topic is by Davis et al.,55 whichcompiles and synthesizes information from over 200 publications Clare46 adds substantial andcomprehensive 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 discoveringnatural product antifoulants The first step is showing that an organic extract of some kind oforganism prevents settlement of a target species.66 Then, depending upon the potency, stability, andmaturity of chemical techniques for determining that particular class of natural product and theavailability of the techniques to the investigators, there are varying levels of success in determiningthe structure of the active molecule Initially, steroids,67 terpenes,44,51 phenolics,68 bromonated9064_ch17/fm Page 547 Tuesday, April 24, 2001 5:29 AM

548 Marine Chemical Ecology

TABLE 17.1

Some Reports of Natural Product Antifoulants from 1995–Present

Extract or Parent Compound

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

Bryozoan

—

—

75

Cytotoxicity

4.3–5 µ g/ml 2.1–3.4 µ g/ml

152

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

Ascidian (Promoter)

15–19 µ g/ml 2.5 µ g/ml

153

Bacteria

0.5–5 µ g/ml

—

156

Inhibition

Induction

Metamorphosis Inducer

9064_ch17/fm Page 548 Tuesday, April 24, 2001 5:29 AM

Antifoulants and Coatings 549

hydrocarbons,20,59 brominated tyrosine derivatives,69 and saponins70 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, Clare46 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

species76 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 untenable79 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

Metamorphosis Inhibitor

TABLE 17.1 (CONTINUED)

Some Reports of Natural Product Antifoulants from 1995–Present

Extract or Parent Compound

9064_ch17/fm Page 549 Tuesday, April 24, 2001 5:29 AM

550 Marine Chemical Ecology

III NATURAL PRODUCTS ANTIFOULANTS

A B IOLOGICAL T ARGETS AND A B RIEF H ISTORY OF N ATURAL P RODUCT

A NTIFOULING S TUDIES

As the natural products antifouling field matures, the nature and diversity of organisms used infouling assays is increasing, as is our understanding of the complexity of the biological processesinvolved Initially, researchers assumed that fouling was a successional process and that microbialfouling 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 paper85specifically addressed settlement by polychaetes, and the assumption of successional foulingappears correct for many polychaetes.17,85,87,88 Corpe86 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 Astatement to this effect can be found in virtually every report on control of microfouling far into

89stimulate settlement of bryozoans on some surfaces90 and settlement of bivalves like oysters91,92 onothers, and that bacterial films may have a variety of effects, ranging from nothing to inhibitingbarnacle 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 bacterialfilms have a major impact on most fouling organisms,98,99 but preventing microfouling will notprevent 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, andchemical surface characteristics mediate the settlement of many common macrofoulers.9,21,28,29Finally, at all levels of fouling, there are some organisms that settle like dust on all surfaces.8,21,55The major early exception to using microfoulers instead of macrofouling organisms in foulingstudies was the use of settlement stage barnacles Barnacles were targeted initially because theywere recognized as central to the failing of antifouling coatings.71,72,100 Barnacles are dominantmembers 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 arelatively robust adhesive

Since cyprid glue contains barnacle settlement pheromones,101,102 even if the first wave oflarvae dies, the glue and tests left behind stimulate settlement of other barnacle larvae while atthe same time acting as a barrier to toxin diffusion If the relatively vulnerable cyprid surviveslong enough to settle and metamorphose, the newly recruited “pin head” barnacle has its ownlarval glue and uncalcified base plate to act as barriers to diffusion from below The top of the pinhead barnacle is elevated approximately 500 µm over the surface, just high enough to enable thebarnacle 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 boundarylayer 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 settlementpheromones102,105,106 thereby furthering recruitment

The result is that barnacles are often the first macrofoulers to colonize a protected surface.100Once growth of barnacles on toxic metal coatings is initiated, the surface rapidly fails becausebarnacles act as nontoxic platforms for other organisms As a result of these kinds of fieldevidence, the U.S Navy supported basic barnacle research for almost four decades, and barnacleswere 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 UniversityMarine Laboratory.47

At Duke in the early 1980s, mass cultured barnacle larvae were used as the target organism inbioassay directed studies of natural inhibitors of fouling Approximately 70% of the marine organisms

) There is also ample evidence that bacterial films canthe 1990s (see for example, Readman

Antifoulants and Coatings 551

in the temperate to subtropical estuarine communities around the Marine Laboratory were found tocontain compounds with readily detectable antibarnacle settlement activity Many compounds areextremely 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 complexorganisms like barnacles is a logical consequence of the complexity of the biochemical pathwayscontrolling metamorphosis There is evidence that these pathways begin with chemoreceptors cou-pled to neuronal, hormonal, and metabolic control processes through classic amplification cascadesthat include second messengers20,22,88,102 that are stimulated by changes in ion permeability.109

B M ECHANISM OF A CTION OF N ATURAL P RODUCT A NTIFOULANTS

The mechanisms of action of most compounds is not obvious from their structures Althoughcertain major classes of molecules, such as steroids, are recognized to have known functions,molecules are usually multifunctional Examination of the structures presented illustrate thispoint Compound 17.1 is a sodium/potassium ATPase inhibitor and a potent natural producttoxin.110 Compound 17.2 is a component of the fragrance of peaches and apricots and somehowanesthetizes 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 thatCompound 17.4 has many of the characteristics of sesquiterpene antifoulants, which act via anunknown mechanism, and hazard that Compounds 17.5–17.9 might function through interactionwith a surface or act like detergents

O HO

N O

H

OH O

Bufalin 10pg/mL (17.1)

552 Marine Chemical Ecology

As molecules increase in complexity, the number of possibilities for mechanism of action alsoincrease For example, Structure 17.10 inhibits a specific kinase and specific cellular secondmessenger.111 In contrast, Structure 17.11 is specifically related to vitamin B2,112 whileStructure 17.12, a steroid peroxide,113 is, in the imagination of this author, a molecule likely toimpact 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

O O

Bromophenol (17.8)

NaO3SO NaO3SO

OSO3Na

NHCHO H

H O

Sterol Peroxide (17.12)

Antifoulants and Coatings 553

effects of these molecules have not been studied Anyone seriously considering commercialapplications of such molecules should simultaneously consider environmental consequences.Potentially as confounding and just as dangerous a pitfall is the assumption that a knownpathway is the mechanism of action for a known molecule Structures 17.13–17.17 all function invertebrate and invertebrate biogenic amine-mediated neurotransmission These molecules also altersettlement and metamorphosis of larvae of fouling organisms Although these molecules may ormay not have commercial potential as antifouling agents, they do point to the possibility thatpharmaceuticals and natural products that specifically alter biogenic amine-mediated pathways may

be candidates for antifouling technology One need only consider that drugs used to alter moods

in humans impact biogenic amine pathways to grasp the magnitude of the issues that must beconsidered for developing organic alternatives to the use of toxic metals On one hand, the metalshave clear environmental impact, but on the other hand, their impact on food supplies and humanhealth is understood This is not the case for organic additives

Historically, funding sources that have supported natural product antifoulant studies have notfunded inquiry into detailed mechanisms of action of a particular candidate compound The logichas been that knowledge of the mechanism is not important for developing a product This ignoresmany significant issues, such as improvement of performance by quantitative structure–functionstudies,80–82,114,115 prediction of effects on nontarget species, chronic long-term studies, and fatesand effects of bioactive additives A few examples resulting from similar approaches to biocidesinclude dichloro-diphenyl-trichloroethane (DDT), mercury, lead arsenic, cadmium, and TBT A

H N

OH HO

Isoproteranol (17.17)

OH

N

N HN O

NH2

NH2

OH HO

Norepinephrine (17.14)

OH

NH2

OH HO

DOPA (17.13)

554 Marine Chemical Ecology

consequence of this approach is that major environmental damage and monetary commitment areinvolved before problems are discovered and understood Many researchers hope that a change inapproach is forthcoming

Those intimately familiar with bioassays have pushed these assays to their limits with respect

to determining mechanisms of action at the whole animal level Three obvious mechanisms areeasy to discern Two mechanisms, internal to the larvae, are toxicity and anesthesia.84,107 The thirdmechanism is external to the larva and involves preventing settlement by molecules adsorbing tothe surface and changing its characteristics.29

Adsorption onto a surface may also impact settling organisms by altering delivery of abioactive compound.47,84 For example, if toxicity assays are performed using both presettlementswimming barnacle larvae with hydrophyllic surfaces (nauplii) and the settlement-stage barnaclelarvae with a lipophylic surface, the results depend on the chemical surface properties of the testcontainer Assays in glass show that both kinds of larvae are susceptible to similar levels oftoxin However, assays in polystyrene containers suggest that nauplii are more resistant to TBT.This result is obtained because TBT is lipophylic and adsorbs from solution onto plastic Theconcentration of TBT is reduced in solution, where the nauplii are exposed and concentrated onthe polystyrene container surface, where it partitions from the surface into the cyprid larvaethrough its lipophylic surface

Since little or no attention is paid to the detailed mechanisms of action of existing additives,many commercial coatings, even those that are advertised as nontoxic, contain potent nonspecificbiocides.47 Performance of coatings is enhanced by adding oils116 and surface-active agents Theauthor’s research group at Duke was surprised to discover, for example, that silicone detergentswere considered nontoxic This is surprising insofar as a variety of surfactants and detergents areroutinely used as insecticides and bacteriocides When tests were conducted with silicone detergents,these detergents were found more toxic to larvae than copper on a weight basis (Rittschof, Bonaven-tura, Gerhart, and Clare, unpublished data) Also surprising was the revelation that many dimethylsilicone coatings are polymerized using organotin catalysts in sufficient concentration (≈ 250 µg/cm2

in experimental coatings tested) to render the coating initially toxic.47 Dimethylsilane coatingspolymerized by dibutyltin have been declared exempt from the Federal Insecticide Fungacide andRodentacide Act (FIRFA) by the EPA Office of Pesticides

In summary, natural products have a few known general mechanisms of action These includetoxins, inhibitors of growth, surface-energy modifiers, nervous pathway interference (anesthetics,including nitric oxide synthase inhibitors and neurotransmitter blockers), inhibitors of attachment,inhibitors of metamorphosis, and repellants.46,55,117,118 It is likely that many compounds have multiplemechanisms of action

C T OXIC M ECHANISMS

Should the final outcome of antifouling research be that toxins are necessary components ofantifouling compounds, some biological compounds are among the most toxic known For example,Donald J Gerhart, then at Duke University Marine Laboratory, considered the antisettlement activityand toxicity of a set of natural toxins including a potent sodium/potassium ATPase inhibitorBufalin.48 Bufalin, the most potent natural product tested, was over 100 times more toxic than TBTand over 6000 times more potent than TBT with respect to antisettlement activity The absence of

a strict correlation between toxicity and antisettlement activity can be partially attributed to therelative solubility of the compounds in seawater and differences in the uptake kinetics betweendifferent life stages However, the major difference can be attributed to the relationship betweenthe speed at which the toxin kills and the speed at which metamorphosis occurs For slow-actingtoxins such as metals, settlement, and metamorphosis often precede death.107