WO1997009464A2

WO1997009464A2 - Isonitrile anti-fouling agents

Info

Publication number: WO1997009464A2
Application number: PCT/US1996/014656
Authority: WO
Inventors: Mark R. Hagadone
Original assignee: Synthetic Technology Corporation
Priority date: 1995-08-31
Filing date: 1996-08-30
Publication date: 1997-03-13
Also published as: WO1997009464A3; AU6976096A

Abstract

A method is disclosed for preventing the accumulation of aquatic organisms on structures continuously exposed to water. Naturally-occurring isonitrile compounds, or analogues or hydrolysis by-products thereof such as the corresponding formamide, isocyanate or isothiocyanate, are applied to surfaces that will be continuously exposed to an aquatic environment. The isonitrile compounds prevent the settling and attachment of aquatic organisms. The isonitrile compounds comprise at least one isonitrile group attached to a hydrocarbon moiety consisting of at least six carbon atoms. The isonitrile compounds can be formulated in a variety of forms, including as paints, solutions or emulsions.

Description

ISONITRILE ANTI-FOULING AGENTS

Field ofthe Invention This invention relates to methods for preventing the attachment of aquatic organisms to surfaces which are submerged for extensive periods of time in water. More particularly, this invention relates to the treatment of submerged surfaces with isonitrile, formamide, isothiocyanate and isocyanate anti-fouling agents.

Background ofthe Invention

Structures that are continuously submerged in water, such as the hulls of ships, water intake pipes and the foundations of oil rigs, become encrusted with a variety of aquatic organisms such as Balanus, Anomia, Enteromorpha, Hydroides, Dreissenia and others. The attachment of these organisms to these structures causes economic damage in various ways: for example, attachment to the hulls of ships reduces fuel efficiency and causes the loss of profitable sailing time because of the need to clean the hulls. Similarly, the attachment of these organisms to cooling water equipment decreases heat conductivity which eventually reduces the cooling power of the equipment and drives up costs.

A variety of agents useful for controlling fouling organisms in fresh water or sea water have been used to prevent the attachment and overgrowth of these organisms. Copper compounds and organotin compounds are known to be active anti-fouling agents, and are still extensively used today. Anti-fouling agents in the form of a paint can contain 10-50% by weight ofthe active ingredient and can be used to paint surfaces such as the hulls of ships. The paint prevents attachment and growth of fouling organisms by continuously releasing anti-fouling agents underwater. The disadvantage of conventional anti-fouling agents is that they are persistent in the environment, are acutely toxic and bioaccumulate. Conventional anti-fouling agents therefore cause considerable damage to the aquatic environment and eventually enter the human food chain.

Environmental problems, such as seas and rivers polluted with heavy metals and toxic compounds, threaten human health and the environment. For example, it is well established that heavy metal compounds, especially organotin compounds that are widely used as anti-fouling agents, accumulate in the human body as a result ofthe consumption offish and shellfish. There is a clear and present need for safe, effective anti-fouling agents.

Summarv ofthe Invention It has now been found that compounds containing an isonitrile group attached to a hydrocarbon moiety of not less than six carbon atoms, or a hydrolysis by-product or analog thereof such as the corresponding formamide, isocyanate or isothiocyanate, are highly effective anti-fouling agents. Many such isonitrile compounds exist in nature as natural defense compounds secreted by marine organisms. These compounds do not, therefore, pose the same risks to human health and to the environment as conventional anti-fouling agents containing heavy metals.

Brief Description ofthe Drawings The foregoing aspects and many of the attendant advantages of this invention will become more readily appreciated in connection with the following detailed description and accompanying drawings, wherein:

FIGURE 1 is a graphic representation of the inhibitory effect of 9-isocyanopupukeanane on the settling of Hydroides elegans larvae on optimally biofilmed substrate;

FIGURE 2A is a graphic representation of the inhibitory effect of the settling of hardfoulers (tubeworms, oysters and encrusting bryozoans) on fiberglass rods coated with a silicon based antifouling paint containing 9-isocyanopupukeanane as described in Example 6;

FIGURE 2B is a graphic representation of the inhibitory effect of the settling of softfoulers (diatoms, hydroids, tunicates and sponges) on fiberglass rods coated with a silicon based antifouling paint containing 9-isocyanopupukeanane as described in Example 6;

FIGURE 3 is a graphic representation of the inhibitory effect ofthe settling of Hydroides elegans on fiberglass rods coated with a silicon based antifouling paint containing 9-isocyanopupukeanane as described in Example 6; FIGURE 4A is a graphic representation of the inhibitory effect of the settling of diatoms on fiberglass rods coated with a silicon based antifouling paint containing 9-isocyanopupukeanane as described in Example 6;

FIGURE 4B is a graphic representation of the inhibitory effect of the settling of arborescent hydroids on fiberglass rods coated with a silicon based antifouling paint containing 9-isocyanopupukeanane as described in Example 6;

FIGURE 5 A is a graphic representation of the inhibitory effect of the settling of bivalves on fiberglass rods coated with a silicon based antifouling paint containing 9-isocyanopupukeanane as described in Example 6; and FIGURE 5B is a graphic representation of the inhibitory effect of the settling of mudtubes (polychaetes and amphipods) on fiberglass rods coated with a silicon based antifouling paint containing 9-isocyanopupukeanane as described in Example 6. FIGURE 6 A is a graphic representation of the inhibitory effect of the settling of hardfoulers (tubeworms, oysters and encrusting bryozoans) on fiberglass rods coated with antifouling coatings as described in Example 8;

FIGURE 6B is a graphic representation of the inhibitory effect of the settling of softfoulers (diatoms, hydroids, tunicates and sponges) on fiberglass rods coated with antifouling coatings as described in Example 8;

FIGURE 7 is a graphic representation of the inhibitory effect of the settling of Hydroides elegans on fiberglass rods coated with antifouling coatings as described in Example 8;

FIGURE 8 A is a graphic representation of the inhibitory effect of the settling of diatoms on fiberglass rods coated with antifouling coatings as described in Example 8; FIGURE 8B is a graphic representation of the inhibitory effect of the settling of arborescent hydroids on fiberglass rods coated with antifouling coatings as described in Example 8.

Detailed Description ofthe Preferred Embodiment It has now been discovered that isonitriles, and/or their hydrolysis by- products, are effective in the environmentally friendly prevention of fouling of submerged structures by aquatic organisms. More particularly, it has now been found that sesquiterpene isonitriles, e.g., 9-isocyanopupukeanane, are highly useful as anti¬ fouling agents. Thus, in accordance with one aspect ofthe present invention, methods are provided for the prevention of attachment of underwater fouling organisms on a surface by treating the surface with an anti-fouling composition comprising at least one isonitrile compound or a hydrolysis by-product thereof. Although the mechanism of attachment of underwater fouling organisms onto surfaces is not fully understood, and the inventor does not intend to be bound by any particular theory, it is presently believed that the isonitrile compounds of the present invention, or the hydrolysis by-products thereof, act by interfering with the health or surface chemistry of the bacterial components of biofilm formed on the surfaces that have been exposed to seawater, which are required for attachment ofthe many aquatic fouling organisms. See, for example, M.G. Hadfield et al., Recent Developments in Biofouling Control, M.R. Thompson et al. Eds., pp. 66-74, Oxford and IBH Pub. Co., New Delhi (1994). Thus, in accordance with another aspect of the invention, methods are provided for the prevention or growth of bacteria or parasites, in vitro or in vivo, by treating the bacteria or parasites with an effective amount of an antimicrobial composition comprising at least one isonitrile compound or a hydrolysis by-product thereof.

The term "anti-fouling" is used herein to refer to any isonitrile compound that prevents the attachment and growth of aquatic organisms to structures that are exposed to an aquatic environment for prolonged periods of time.

Isonitriles (isocyanides) are a class of naturally-occurring, nitrogen containing, organic compounds that are often biologically active. The terminal carbon is attached to the rest of the molecule via the relatively stable nitrogen atom, rather than the reverse as in nitriles. The isonitrile functionality is labile on exposure to water, and slowly forms various by-products, including the corresponding formamide, in aqueous environments. Therefore, as used herein the term "isonitrile" is intended to include the isonitrile compounds themselves, as well as the hydrolysis by-products thereof, such as formamides, that are formed as a chemical or biological consequence in the aqueous environment of their use as described herein. The term "isonitrile" is also intended to include the analogs ofthe isonitrile compounds, such as the corresponding isocyanates and isothiocyanates. Isonitriles useful in the present invention preferably have at least one isonitrile group attached to a hydrocarbon moiety comprising at least six carbon atoms. The isonitrile compounds may be isolated from natural biological material or synthetically produced.

The first naturally occurring isonitrile to be isolated was xanthocillin, an amino acid derivative found in the mold Penicillium notatum (W. Rothe, Chemical Abstracts 44 8063e). Isonitriles are now known to occur in marine organisms, especially in sponges where approximately 140 isocyano compounds have been identified (C.W.J. Chang et al., Topics in Current Chemistry 167:34-74 (1993)). Naturally occurring marine isonitriles are either sesquiterpenes or diterpenes with one, two or three isocyano (or isocyano-related) functions. The non-polar hydrocarbon moiety is usually cyclic, but acyclic isonitrile diterpenoids are known (B.J. Burreson et al., Tetrahedron 31:2015-2018 (1975)).

Examples of naturally-occurring isonitrile compounds that can be used in the present invention include, but are not limited to: axisonitrile-1, axisonitrile-4, acant- hellin-1, 6α-isocyano-5α-H,7α-H,10-α-eudesm-4(14)ene, stylotelline, 11-isocyano- 7β-H-eudesm-5-ene, (-)-10-isocyano-4-amorphene, 4α-isocyano-9-amorphene, 9-iso- cyanopupukeanane, 9-epi-9-isocyanopupukeanane, 3-isocyanotheonellin, axiso- nitrile-2, axisonitrile-3, cavernoisonitrile, 10-α-isocyano-4-amorphene, 7-isocyano- 7,8-dihydro-α-bisabolene, epipolasin-A epipolasin-B, 3-isocyanotheonellin, 8,15- diisocyano-1 l(20)-amphilectene, 8-isocyano-l(12)-cycloamphilectene, 8-isocyano- 10(14)-amphilectadiene, kalihinols A-J, kalihinols X-Z, kalihinene, isokalihinol B, iso- kalihinol F, 10α-isocyanoalloaromadendrane,l-isocyanoaromadendrane, 2-isocyano- pupukeanane, 2-isocyanoallopupukeanane, 9-isocyanoneopupukeanane, 3-isocyano- theonellin, 7-isocyano-7,8-dihydro-α-bisabolene, diisocyanodociane, 8, 15-diisocyano- 11 (20)-amphilectene, 7-isocyano- 11 (20), 14-epiamphilectadiene, 7-isocyano- 11 (20), 15-epiamphilectadiene, 7, 15-diisocyano- 11 (20)-epiamphilectene, 7, 15-diiso- cyanodociane, 8-isocyano- 10-cycloamphilectene, 8-isocyano- 1 ,( 12)-cycloamphi- lectene, 7-isocyano-l 1-cycloamphilectene, 7-isocyano-l-cycloamphilectene, 8-isocyano-10,14-amphilectadiene, 7-isocyanoneoamphilecta-l l,15-diene, caverno¬ isonitrile.

All of the isonitrile compounds described above are found in sponges and can be obtained by extraction and purification from these organisms. Typically, the whole organism is extracted with aqueous alcohol, although lyophilized material may be used. The crude extract is then subjected to the Kupchan purification scheme, which partitions constituents on the basis of polarity by means of sequential solvent extractions of the crude aqueous extract (S.M. Kupchan et al., J. Org. Chem. 38:178-179 (1973)). Examples of useful solvents are hexane, carbon tetrachloride and chloroform. In those cases where the amount of starting material is limited, or non-aqueous conditions are preferred, blending ofthe lyophilized sponge with organic solvents is employed. Solvent-partitioned mixtures are fractionated further by one or more column chromatography steps, for example utilizing Sephadex chromatography, high performance liquid chromatography and silica gel chromatography. The biological activity ofthe column fractions can be assayed at each purification step. For example, 9-isocyanopupukeanane can be isolated from the sponge,

Ciocalypta sp. by ethanol extraction of the dry sponge, followed by thin layer chromatography of the oily extract on silica gel with hexane as a solvent (B . Burreson et al., J. Am. Chem. Soc. 97:4763-4764 (1975)). 9-Isocyanopupukea- nane can also be isolated from the nudibranch Phyllidia varicosa, which feeds upon Ciocalypta sp. and sequesters the isonitrile. 9-Isocyanopupukeanane can be purified from Phyllidia by collecting and vacuum distilling the mucus secreted by Phyllidia, followed by extraction ofthe distillate with methylene chloride, then separation by thin layer chromatography on alumina with a mixture of methylene chloride and hexane as the solvent (B.J. Burreson et al., vide supra).

The biological source material of many of the naturally-occurring isonitriles is frequently limiting, so it will often be preferable to chemically synthesize these compounds when large quantities are required, such as in the practice of the present invention. The synthesis of many naturally-occurring isonitrile compounds has been achieved, including the synthesis of 9-isocyanopupukeanane (E. J. Corey et al., J. Am. Chem. Soc. 101:1608 (1979); H. Yamamoto et al., J. Am. Chem. Soc. 101: 1609 (1979)). It may also be preferable to chemically synthesize simpler analogues of naturally occurring isonitriles, such as 9-isocyanopupukeanane, that comprise at least one isocyano group and either some of the carbon framework of the natural product, or a linear hydrocarbon moiety of at least six carbons. For purposes of illustration, representative embodiments of synthetically produced isonitriles of the present invention are shown below, including 9-isocyanopupukeanane (structure 1) and representative synthetic analogues having the structures 2, 3, 4 and 5:

1 2 3 4 5

The compounds of the present invention may be synthesized in accordance with several general synthetic methods that have been reported. For example, the hydrocarbon part of the molecule can be 1 -adamantyl or 2-adamantyl . The primary amines of these compounds can be reacted with chloroform to yield the corresponding isonitriles as shown below (Weber et al., Tetrahedron Letters 17:1637-1640 (1972)):

R-NH₂ + CHC1₃ R-NC

Where R is selected from, for example:

Alternatively, it may be preferable to utilize the reaction between cyanide ions and an appropriate alkyl halide as shown below (Friedrich et al., The Chemistry ofthe Cyano Group, Rappaport, Ed., pp. 77-86, Interscience Publications, New York (1970):

R-Hal + AgCN R-NC

Where R is selected from, for example:

The isonitrile compounds ofthe present invention can also be prepared by the elimination of water from N-alkylformamides with phosgene and a tertiary amine, as shown below:

R-NH-C(=O)-H R-NC

Where R is selected from, for example:

Other reagents including TsCl in quinoline, POCI3 in pyridine and chloro- dimethylformiminium chloride are also effective. The N-alkylformamides can be prepared from the corresponding amines by formylation with formic acid.

The isonitrile compounds or hydrolysis by-products thereof used as the active ingredient in the present invention may be used alone or in combination with other anti-fouling agents. The anti-fouling compounds ofthe present invention may be used by formulating the agents in numerous liquid forms, including, without limitation, as a paint, a solution and as an emulsion. Formulation is carried out by routine means known to the art. For example, the reagents of the present invention may be used as an antifouling coating or paint. An anti-fouling coating or paint is prepared by formulating one or more of the active ingredients described above with other components described below. The anti-fouling coating or paint can then be applied to ship hulls or to other underwater structures. The anti-fouling coating or paint comprises the active ingredient of the present invention, that is, a naturally-occurring or synthetic isonitrile compound, or analog thereof, and film-forming coating ingredients, including solvents which are selected according to use, extender pigments, coloring pigments and additives. Suitable film-forming coating ingredients include, for example, marine paint coatings, silicone rubber resin, chlorinated rubber resin, vinyl acetate resin, acrylic resin and natural resin. In one presently preferred embodiment of the invention, the film-forming coatings may comprise polysiloxanes, substituted polysiloxanes, modified polysiloxanes and/or copolymers thereof, such as those described in U.S. Patent Nos. 4,080,190, 4,861,670 and 5,258,225. In a presently particularly preferred embodiment, the film-forming coatings of the invention may comprise a poly(dimethyl-siloxane) polymer such as that marketed as RTV11 by General Electric Research Corporation, Schenectedy, New York, U.S.A. The active isonitrile is typically formulated 0.001-25% by weight based on the weight ofthe anti-fouling paint, preferably 0.01-10%. In an alternative embodiment of the present invention, the active anti-fouling isonitrile functionality may be incorporated directly into the polymer chain ofthe anti¬ fouling paint, by covalent bonding to the polymer. In a presently preferred embodiment of this aspect of the invention, the film-forming coatings ofthe invention comprise an isonitrile-containing polysiioxane, such as a polysiioxane having the formula:

wherein R-_^ and R₂ are independently selected from loweralkyls having from 1 to 10 carbon atoms, e.g., methyl, ethyl, propyl, butyl, pentyl, etc.; aryl, e.g., phenyl, tolyl, xylyl, etc.; cycloalkyl, e.g., cyclohexyl, cycloheptyl, etc.; or aralkyl, e.g., benzyl, phenylethyl, etc.; either alone or substituted with one or more isonitrile groups; and X and Y represent relative percentages of the number of respective moieties in the polysiioxane, with X being less than about 10%, more preferably less than about 5% and most preferably less than about 1% of the polysiioxane, and Y being preferably greater than or equal to 90%, more preferably greater than or equal to about 95% and most preferably equal to about 99% ofthe polysilane. Representative polysiloxanes of this group include, for example, coatings ofthe formulas:

When the components ofthe present invention are used as a solution, an anti¬ fouling solution is prepared by formulating the active ingredients with film-forming ingredients as described above and by dissolving the mixture in solvents. Film- forming ingredients used in the anti-fouling solution include chlorinated rubber resin, vinyl acetate resin, acrylic resin and natural resin. Solvents include toluene, xylene, cumene, methylisobutylketone, ethyl acetate and methanol. Additives, such as a plasticizer, can be added to the anti-fouling solution if necessary. The active ingredient is typically formulated 0.001-70% by weight based on the weight of an anti-fouling solution, preferably 0.01-30%. The anti-fouling solution can be applied, for example, to farming nets and sea nets in order to prevent overgrowth of fouling organisms.

When the agent is employed as an emulsion, an anti-fouling solution is prepared according to the conventional method in the art, by dissolving active ingredients described above in solvents and by adding surfactants to the mixture. Surfactants include those typically used in the art. When the reagents of the present invention are used as an emulsion, the active ingredient is typically formulated 0.001-50% by weight based on the weight of the anti-fouling emulsion, preferably 0.01-40%. The anti-fouling emulsion can also be applied, for example, to farming nets and sea nets in order to prevent overgrowth of fouling organisms.

EXAMPLES The present invention will be more readily understood with reference to the following example. However, this example is intended to illustrate the present invention and is not to be construed as limiting the scope ofthe invention. EXAMPLE 1 Effect of 9-isocyanopupukeanane on the Settling of Hydroides elegans Larvae

A laboratory biological assay was used to determine if the compound, 9-iso- cyanopupukeanane (ICP), was effective at delaying and/or inhibiting settlement ofthe sessile, calcareous, polychaete tubeworm Hydroides elegans. A stock solution of

9-isocyanopupukeanane was prepared at a concentration of 1 mg/1 ml by dissolving the 9-isocyanopupukeanane in methanol. A total amount of 1 μg, 10 μg and 100 μg of the 9-isocyanopupukeanane in methanol solvent was then applied to small filter paper disks. The solvent was then evaporated to dryness. As controls, filter paper disks were treated with methanol alone, or were left untreated (blanks).

For each treatment, 10 replicate filter paper disks were placed into separate 60 x 15 mm, sterile, plastic petri dishes in 15 ml of 0.45 μ Millipore-filtered seawater. After one hour, 150 larvae oi Hydroides elegans, competent to settle, were added to each dish along with 0.40 ml of phytoplankton (larval food). A biofilmed 2 cm square of plastic mesh (Vexar™) was provided in each dish as an optimal settlement surface, and settlement of this surface in each treatment was compared. The Vexar mesh was biofilmed by floating for 3 days in a water table with a well developed natural microbial community. The number of larval settlers was counted after 24 hours, and again at seven days. After 7 days all individuals had either settled or died. Two factors related to larval settlement were considered: larval toxicity and larval settlement rate. Toxicity of each compound was assessed by comparing the total number of settlers in the blank treatment with the total number of settlers in all other treatments. Only a mean of 2.8 larvae (of the 150 added to each dish) survived and settled in the 100 μg treatment. Thus, this treatment was considered to be toxic and was not used further. Mortality was negligible in all other treatments.

The settlement rate results for non-toxic treatments are presented in Table 1, below, and in FIGURE 1. The settlement rate is expressed as the proportion of larvae that settled in the initial 24 hour time period, divided by the total number of settlers. Statistically significant differences at the p<0.05 level (95% confidence that the two means are different) were then determined using a Kruskal- Wallis Test. Table 1 Proportion oi Hydroides elegans larvae settling in 24 hours:

Treatment Mean ± SD

Blank 64.62 ± 5.69

Methanol 64.85 ± 8.41 l μg lCP 53.08 ± 5.21 lO μg lCP 29.60 ± 4.71

100 μg ICP toxic

Statistical comparisons of the proportion of Hydroides elegans larvae settling in 24 hours are shown in the following Table 2 with significantly different pairs marked with an asterisk (*):

Table 2

Comparison p value

Blank vs. 1 μg ICP 0.1306

Blank vs. 10 μg ICP 0.0009*

Methanol vs. 1 μg ICP 0.0821

Methanol vs. 10 μg ICP 0.0102*

From this bioassay, it was apparent that 9-isocyanopupukeanane was toxic to

Hydroides elegans larvae at the highest concentration tested, and that it significantly delayed settlement at the intermediate concentration. EXAMPLE 2

ICP was presented to Hydroides elegans larvae at several different concentrations in filter paper disks to ascertain the effective concentration range. A stock solution of ICP was prepared at a concentration of 1 mg/ml by dissolving the ICP in methanol. Various concentrations were then prepared by applying the appropriate amount to filter paper disks and the solvent was then evaporated to dryness. As controls, filter paper disks were treated with methanol alone, or were left untreated (blanks). The assays used 0.1 μg, 1.0 μg and 10 μg ICP, tested in two experiments with different batches of larvae. The assays were performed in 5 X 10 mm polystyrene petri dishes filled with 5 ml 0.45 μ Millipore-filtered seawater. Approximately 50 competent larvae (5 days old) were added to each dish, along with an approximately 2 cm square of biofilmed plastic mesh (Vexar™) as the settlement substrate. The Vexar mesh had been biofilmed by floating for 3 days in a seawater table with a well developed natural microbial community. One treatment had only biofilmed Vexar as a positive settlement control. Five replicate dishes of each treatment were used and the results are shown in the following tables. The number of larval settlers was counted in each dish after 24 hours as number of tubes present, and total number of larvae was counted at 7 days. Settlement was expressed as percent ofthe total in each dish. The proportions of settled larvae are shown in the following Table 3:

Table 3 Mean Proportion of Hydroides elegans Larvae Settled (%)

Treatment Day Mean ± SD

Blank 0.42 ± 0.07 Methanol 0.55 ± 0.08 No disk added 0.48 ± 0.21 0.1 μg lCP 0.44 ± 0.10 1.0 μg ICP 0.39 ± 0.09 10 μg ICP 0.41 ± 0.10

Blank 7 0.98 ± 0.04 Methanol 7 0.98 ± 0.04 No disk added 7 0.99 ± 0.02 0.1 μg ICP 7 0.98 ± 0.02 1.0 μg ICP 7 0.95 ± 0.06 lO μg lCP 7 0.88 ± 0.05

The differences in settlement between treatments was evaluated with a non- parametric rank test, using the probability value of p<0.05 in a Kruskal-Wallis Chi- square approximation to indicate significance. Blank and methanol treatments were compared to the plain biofilm treatment, and the ICP treatments were compared to the methanol treatment. The results are shown in the following Table 4:

Table 4 Evaluation of Settlement Differences

Day of Proportion Proportion

Comparison Count Settled Rank Swimming Rank

Blank vs Plain Vexar (wt) 1 0.8340 0.2948

7 0.5205 0.5205

Methanol vs Plain Vexar (wt) 1 0.6015 1.0000

7 0.2204 0.1060

0.1 μg ICP vs Methanol 1 0.1172 0.3457

7 0.5970 0.6623

1.0 μg ICP vs Methanol 1 *0.0472 methanol> 0.2948

7 0.5970 1.0000

10 μg ICP vs Methanol 1 *0.0283 methanol> 0.1425

7 *0.0090 methanol> *0.0080* ICP10>

Both experiments with 0.1, 1.0 and 10 μg of ICP show significant reduction in the proportion of larvae settled in the 10 μg ICP treatment. In the first experiment larvae were added to dishes at roughly the same time as the ICP disk and the biofilmed substrate. Although settlement was inhibited at the highest ICP concentration, many animals did settle and metamorphose. In the second experiment ICP disks and biofilmed substrate were placed into the dishes and allowed to stand 4 hours before larvae were added so that the ICP might have an effect on the biofilm before exposing larvae to it, in line with our hypothesis that ICP may act as an antibiotic. The reduction of settlement in the 10 μg ICP treatment was much stronger in this experiment, as shown in the following Table 5:

Table 5 Mean Proportion of Hydroides elegans Larvae Settled (%)

Treatment Day Mean ± SD

Blank 0.46 ± 0.09

Methanol 0.58 ± 0.23

No disk added 0.47 ± 0.12

0.1 μg lCP 0.47 ± 0.18

1.0 μg ICP 0.48 ± 0.18

10 μg ICP 0.17 ± 0.09

Blank 2 0.83 ± 0.21

Methanol 2 0.83 ± 0.13

No disk added 2 0.85 ± 0.18

0.1 μg lCP 2 0.77 ± 0.22

1.0 μg ICP 2 0.73 ± 0.18 lO μg lCP 2 0.37 ± 0.11

Blank 7 0.96 ± 0.03

Methanol 7 0.98 ± 0.03

No disk added 7 0.99 ± 0.03

0.1 μg ICP 7 0.88 ± 0.24

1.0 μg ICP 7 0.92 ± 0.06

10 μg ICP 7 0.56 ± 0.11

EXAMPLE 3

ICP was presented to Hydroides elegans larvae at several different concentrations in filter paper disks to attempt to define the effective concentration range. As in Examples 1 and 2, stock solution of ICP was prepared at a concentration of 1 mg/ml by dissolving the ICP in methanol. Various concentrations were then prepared by applying the appropriate amount (0.5 μg, 5 μg or 50 μg ICP) to filter paper disks and the solvent was then evaporated to dryness. As controls, filter paper disks were treated with methanol alone, or were left untreated (blanks). The assays were performed in 35 X 10 mm polystyrene petri dishes filled with 5 ml 0.45 μ Millipore-filtered seawater. Approximately 50 competent larvae (5 days old) were added to each dish, along with a small rectangle of biofilmed plastic mesh (Vexar™) as a settlement substrate. The Vexar strips were biofilmed by floating them for at least 3 days in a water table with a well developed natural microbial community. Previous experiments had shown this to be sufficient to induce settlement in these polychaete larvae, which will not settle and metamorphose on a clean surface. One treatment had only biofilmed Vexar as a positive control to check for settlement competence of the larvae. Five replicate dishes of each treatment were used in each trial. Settlement was counted in each dish after 24 hours as number of tubes present, and total settlement was counted at 7 days, at which time metamorphosis is usually near 100% in the positive control treatments. Settlement was expressed as a proportion ofthe total in each dish. The results are shown in the following Table 6:

Table 6 Mean Proportion of Hydroides elegans Larvae Settled

Replicate 1 Replicate 2 Replicate 3

Treatment Day 1 Day 7 Day 1 Day 7 Day 1 Day 7

Vexar 0.94 ± 0.07 1.00 0.81 ± 0.15 0.99 ± 0.02 0.67 ± 0.23 1.00

Methanol 0.88 ± 0.11 0.98 ± 0.01 0.68 ± 0.14 1.00 0.71 ± 0.05 1.00

Blank 0.84 ± 0.17 0.99 ± 0.01 0.84 ± 0.15 1.00 0.53 ± 0.18 1.00

0.5 μg 0.92 ± 0.10 0.99 ± 0.01 0.90 ± 0.11 1.00 0.65 ± 0.26 1.00

5 μg 0.72 ± 0.16 0.95 ± 0.04 0.54 ± 0.16 0.90 ± 0.12 0.56 ± 0.21 1.00

50 μg 0.78 ± 0.10 0.97 ± 0.03 0.73 ± 0.07 0.97 ± 0.06 0.51 ± 0.26 1.00

The antibiotic effects of ICP were tested by exposing pure strains of marine bacteria grown on marine agar to 5, 25, 50 and 100 μg concentrations of ICP presented on filter paper disks, with the same controls (methanol and blank) as above. Zones of clearing in the homogeneous bacterial growth indicate an inhibitory effect. Three replicate dishes were used for each bacteria. Again, ICP was effective at inhibiting the growth of species of Bacillus and Vibrio species, but had no effect on a Pseudomonas species. The Bacillus is an orange pigmented species, and one of the effects noted early in the trial was the reduced amount of pigment in the bacteria that did grow in this dish.

EXAMPLE 4

The procedure of Example 3 was repeated except as follows. Two replicate trials were run with the ICP disk (containing 5.0, 25.0, 50.0 or 100.0 μg ICP) added to dishes with biofilmed Vexar one hour before larvae were added. The results are shown in Table 7:

Table 7

The effects of ICP at concentrations of 5, 25, 50 and 100 μg on settlement of

Hydroides elegans larvae in two replicate trials, with five dishes per treatment in each trial

Mean Proportion Settled

Replicate 1 Replicate 2

Treatment Day 1 Day 7 Day 1 Day 7

Vexar 0.68 ± 0.18 1.00 0.89 ± 0.26 0.97 ± 0.02

Blank 0.81 ± 0.13 1.00 0.77 ± 0.10 0.98 ± 0.02

Methanol 0.65 ± 0.24 1.00 0.77 ± 0.11 0.98 ± 0.04

5.0 μg 0.73 ± 0.07 1.00 0.79 ± 0.07 0.98 ± 0.03

25.0 μg 0.80 ± 0.14 0.97 ± 0.05 0.74 ± 0.15 0.95 ± 0.03 50 μg 0.71 ± 0.06 0.98 ± 0.04 0.71 ± 0.10 0.94 ± 0.06

100.0 μg 0.62 ± 0.19 0.92 ± 0.10* 0.76 ± 0.14 0.98 ± 0.04 * significantly different from the methanol control (p<0.05)

Another trial tested the effect of adding the ICP disks 24 hours before larvae were placed into the dishes, giving the compound a longer time to interact with the biofilmed surface. The results are shown in Table 8:

Table 8 The effects of ICP at concentrations of 5, 25, 50 and 100 μg on settlement of

H. elegans larvae where water-table vexar was soaked 24 hours with ICP before larvae were added; four dishes per treatment

Mean Proportion Settled

Treatment Day 1 Day 3 Day 7

Vexar 0.46 ± 0.23 0.84 ± 0.29 0.78 ± 0.16

Blank 0.53 ± 0.15 0.72 ± 0.20 0.93 ± 0.07

Methanol 0.56 ± 0.15 0.83 ± 0.20 0.88 ± 0.11

0.1 μg 0.38 ± 0.29 0.67 ± 0.22 0.83 ± 0.12

1 μg 0.64 ± 0.14 0.93 ± 0.21 0.98 ± 0.03

5 μg 0.34 ± 0.07* 0.58 ± 0.22* 0.83 ± 0.09*

10 μg 0.50 ± 0.31 0.74 ± 0.24 0.76 ± 0.27

50 μg 0.41 ± 0.04 0.65 ± 0.20* 0.80 ± 0.13

100 μg 0.31 ± 0.12* 0.58 ± 0.14* 0.60 ± 0.17*

* significantly different from the methanol control (p<0.05)

Bacterially enhanced biofilm was prepared by culturing Vexar strips for 24 hours with sea water from the Kewalo lab water-table enriched with dilute (1:4) marine nutrient broth for bacteria. These strips were then rinsed and placed into individual bioassay petri dishes with ICP disks for 24 hours before adding larvae. The results are shown in Table 9: Table 9

Effects of ICP on settlement on bacteria enhanced biofilm (b). Vexar strips cultured 24 hours in water-table seawater with dilute marina broth (1:4), rinsed, then soaked 24 hours with ICP disks before adding larvae.

Mean Proportion Settled

Treatment Day 1 Day 7

Water-table vexar 0.84 ± 0.12 0.99 ± 0.02 Vexar-b 0.57 ± 0.22 0.82 ± 0.08*

Blank-b 0.31 ± 0.20 0.73 ± 0.10

Methanol-b 0.61 ± 0.12 0.82 ± 0.16

5.0 μg-b** 0.21 ± 0.05 0.32 ± 0.06

25.0 μg-b** 0.24 ± 0.14 0.35 ± 0.10

50 μg-b** 0.16 ± 0.04 0.27 ± 0.07

100 μg-b** 0.18 ± 0.14 0.35 ± 0.18 *settlement on cultured biofilm significantly less than on water-table biofilmed vexar (p<0.05) ^♦""settlement in ICP trials significantly less than in ICP trials on day 1 and 7 (p<0.05)

The effects of ICP on the biofilming process was investigated by incubating Vexar strips with water-table seawater in the presence of ICP disks for one week. Biofilmed strips were then taken out and put into bioassay dishes with larvae, with fresh filtered seawater and no ICP. The results are shown in Table 10:

Table 10 Effects of ICP on settlement on biofilming process: vexar strips soaked in water- table seawater with ICP disks for one week. Larval settlement tested in filtered seawater without ICP. No significant effect seen.

Mean Proportion Settled

Treatment Day 1 Day 7

Vexar 0.65 ± 0.13 0.93 ± 0.07

Methanol 0.53 ± 0.13 0.77 ± 0.13 5 μg 0.62 ± 0.18 0.86 ± 0.05

10 μg 0.65 ± 0.17 0.92 ± 0.07

50 μg 0.78 ± 0.11 0.97 ± 0.02 The results suggest that ICP at very low concentrations is effective at inhibiting settlement of H. elegans larvae without toxic effects. The greater effectiveness with a longer incubation with ICP prior to adding larvae suggests an interaction with the biofilm. This is supported by the more dramatic reduction in settlement by ICP where the biofilm had a higher density of bacteria. However, the presence of ICP during the biofilming process did not seem to have any effect on the acceptability of these surfaces to settling larvae. EXAMPLE 5

Two different approaches for presenting ICP to larvae were used in order to try to separate the effects of ICP in solution from ICP on a surface. In one method, ICP (and also the crude products of methanol and hexane sponge extracts) was prepared as a sticky solid and coated onto Vexar strips. In another set of trials, solutions of ICP at precise concentrations in filtered seawater were used as the medium in larval settlement assays.

For the coated format, solids from the sponge Ciocalypta were recovered by evaporating 15 ml of crude methanol (polar compounds) or hexane (nonpolar compounds) extracts to dryness using a vacuum pump. Previously purified ICP (from hexane) was also used. Each solid was redissolved in solvent to give a 5000 μg/ml solution (25 mg in 5 ml solvent), then diluted to create 1000 and 100 μg/ml solutions. Plastic mesh (Vexar™) strips (2 X 1 cm) were dipped into these sticky solutions for 30 minutes, then the remaining solution was removed by pipette and dried by vacuum pump. Coated Vexar strips were biofilmed for one week in a seawater table, along with uncoated strips as controls. These biofilmed strips were then presented to Hydroides elegans larvae in using the standard bioassay conditions described in Example 3. The results are shown in Table 11 :

Table 11 Effects of ICP in solution in filtered seawater at concentrations of 0.1, 1.0 and 10 μg/ml on settlement of Hydroides elegans larvae with and without water-table biofilmed vexar

Mean Proportion Settled

Treatment Day 1 Day 7

Plain Vexar 0.18 ± 0.24 0.80 ± 0.18

MethlOO 0.23 ± 0.24 0.57 ± 0.23

MethlOOO 0.28 ± 0.40 0.54 ± 0.25

Meth5000 0.22 ± 0.27 0.50 ± 0.29

HexanelOO 0.20 ± 0.17 0.58 ± 0.22

HexanelOOO 0.21 ± 0.19 0.50 ±0.27*

Hexane5000 0.20 ± 0.22 0.46 ± 0.30*

ICP100 0.19 ± 0.28 0.69 ± 0.17

ICP1000 0.09 ± 0.12 0.44 ± 0.22*

ICP5000 0.08 ± 0.10 0.39 ± 0.25* *significantly reduced settlement (p<0.05)

A downward trend in larval settlement with higher concentrations of sponge extract or pure ICP by the end of the experiment (day 7) is shown in Table 11. In each set, the treatment with the highest concentration of sponge product in the coating showed the lowest larval settlement, with pure ICP giving the strongest reduction of larval settlement overall.

ICP was presented to Hydroides elegans larvae at several different concentrations in solution in 0.22 μ filtered seawater. The isonitrile was dissolved in 0.5 ml methanol, then added to 500 ml seawater to give final concentrations of 10, 1, and 0.1 μg/ml. An identical solution of methanol without ICP was used as a control (0.1% methanol). At 10 μg/ml the solution was cloudy, and slowly cleared with crystals formed on the bottom, indicating that the saturation point for this compound in seawater is something less than 10 μg/ml. Larvae remained alive even in dishes with ICP precipitate on the bottom. The assays were performed as described in Example 3. The results are shown in Table 12:

Table 12 Effects of ICP in solution in filtered seawater at concentrations of 0.1, 1.0, and 10 μg/ml on settlement of Hydroides elegans larvae with and without water-table biofilmed vexar

Mean Proportion Settled With Biofilmed Vexar Without Biofilmed Vexar

Treatment Day 1 Day 7 Day 1 Day 7

Filtered seawater 0.34 ± 0.28 0.82 ± 0.26 0.00 ± 0.00 0.03 ± 0.02 Methanol 0.25 ± 0.38 0.81 ± 0.27 0.00 ± 0.00 0.38 ± 0.34

ICP0.1 0.13 ± 0.17 0.83 ± 0.14 0.00 ± 0.00 0.13 ± 0.13

ICP1 0.14 ± 0.23 0.71 ± 0.25 0.00 ± 0.01 0.13 ± 0.13

ICP10 0.00 ± 0.00* 0.12 ± 0.17* 0.00 ± 0.00 0.00 ± 0.00*

* significantly reduced settlement (p<0.05)

In this trial a set of treatments without biofilmed Vexar tested the effects of the compound alone. At 10 μg/ml settlement was strongly reduced, with larvae ceasing to swim by the second day, but with very few continuing the process of metamorphosis to the completion of a tube. Larvae were still alive after one week in all treatments.

A second trial following the foregoing procedure replicated only the biofilmed Vexar set, with results shown in Table 13: Table 13

Effects of ICP in solution in filtered seawater at concentrations of 0.1, 1, 5 and

10 μg/ml on settlement of Hydroides elegans larvae on water-table biofilmed vexar

Mean Proportion Settled

Treatment Day 1 Day 2 Day 4 Day 7

Filtered seawater 0.23 ± 0.07 0.45 ± 0.12 0.72 ± 0.20 0.96 ± 0.02

Methanol 0.15 ± 0.05 0.32 ± 0.17 0.59 ± 0.35 0.71 ± 0.18

ICP0.1 0.03 ± 0.03* 0.11 ± 0.09* 0.32 ± 0.12* 0.84 ± 0.07

ICP1 O.IO ± O.IO 0.13 ± 0.10* 0.71 ± 0.23 0.71 ± 0.23

ICP5 0.07 ± 0.10 0.07 ± 0.09* 0.17 ± 0.14* 0.54 ± 0.19

ICP10 0.31 ± 0.12 0.09 ± 0.14* 0.15 ± 0.23 0.24 ± 0.16* * significantly reduced settlement (p<0.05)

Larval settlement was dramatically reduced by all concentrations of ICP, with effects lasting through the end ofthe experiment in the higher concentrations.

EXAMPLE 6

A silicone based low surface-energy paint (PDMSO) provided by General Electric Research Corporation was used to coat fiberglass rods (10 cm length) for a one month field trial at Pearl Harbor, Hawaii, United States of America. Pure crystalline ICP was added to the liquid paint, which hardens upon the addition of a catalyst. A IO ppm ICP stock was prepared, mixed well, then diluted with paint for 1 ppm and 0.1 ppm concentrations. Rods were primed, then dipped into the paint after catalyst had been added. Rods coated with paint without ICP was used as a control along with plain fiberglass. The coating was allowed to cure for one week, then the rods were mounted in a random array on 22 cm² PVC pipe frames with Vexar mesh, secured by surgical rubber rings used as grommets. The frames were immersed in Pearl Harbor beneath a raft at Hospital Point. At one week intervals, 5 rods of each type were removed and the settlement of organisms counted. Percent cover of hardfoulers (calcareous tubeworms, oysters and bryozoans) and softfoulers (diatoms, arborescent hydroids, tunicates and sponges) was estimated using a dissecting microscope. Differences in settlement between treatments were analyzed using a Kruskal-Wallis Chi-square approximation. The results are shown in FIGURE 2A for hardfoulers (tubeworms, oysters and encrusting bryozoans), FIGURE 2B for softfoulers (diatoms, hydroids, tunicates and sponges), FIGURE 3 for Hydroides elegans, FIGURE 4A for diatoms, FIGURE 4B for arborescent hydroids, FIGURE 5A for bivalves and FIGURE 5B for mudtubes (polychaetes and amphipods). In FIGURES 2A-5B, FG is fiberglass alone, Cont. is paint control, and ICP0.1, ICP1 and ICP10 represents ICP concentrations of 0.1 μg, 1 μg and 10 μg, respectively.

The field trial indicates that settlement of a variety of organisms is reduced under field conditions, and that ICP is apparently stable in the silicone coating used. The fact that the paint control showed less fouling than the fiberglass rods is typical, and is probably related to the surface characteristic of this very slippery coating.

EXAMPLE 7

The effects of the synthetic isonitrile, 1,1,3,3-tetramethylbutylisonitrile (TMBI) on the settlement of H. elegans larvae was testing in laboratory bioassays in which the isonitrile was present in solution in the assay dish during the experiment. The isonitrile was dissolved in 0.5 ml methanol, then added to 500 ml seawater to give a final concentration of 10 μg/ml. An identical solution of methanol without ICP was used as a control (0.1% methanol). The assays were performed according to the procedure of Example 3 using concentrations of 10 μg/ml, 1 μg/ml and 0.1 μg/ml. Settlement was counted in each dish after 48 hours as number of tubes present, and total settlement was counted at 5 days, at which time metamorphosis was high in the control treatments (no isonitrile). Settlement was expressed as a proportion of the total in each dish. The results are shown in Table 14:

Table 14 Mean Proportion oi Hydroides elegans Larvae Settled

Trial 1 Trial 2

Treatment Day Mean ± SD Mean ± SD

Filtered seawater 1 0.1 ± 0.1 0.11 ± 0.07

Vexar 1 0.68 ± 0.26 0.87 ± 0.16

0.1 μg/ml TMBI 1 0.59 ± 0.14 0.75 ± 0.05 l μg/ml TMBI 1 0.69 ± 0.07 0.77 ± 0.15

10 μg/ml TMBI 1 0.35 ± 0.06 0.68 ± 0.17

Filtered seawater 7 0.02 ± 0.03 0.44 ± 0.27

Vexar 7 1.00 ± 0.00 0.98 ± 0.03

O. l μg/ml TMBI 7 0.99 ± 0.01 l.OO ± O.OO

1 μg/ml TMBI 7 0.98 ± 0.04 1.00 ± 0.00

10 μg/ml TMBI 7 0.80 ± 0.29 0.96 ± 0.07

Although in the first trial larval settlement was significantly reduced (p<0.05), this result was not repeated in the second trial.

The results of trials with tetramethylbutyl isonitrile (TMBI) were not conclusive, but this compound did not seem to be comparable to ICP in its ability to reduce larval settlement. ICP has a longer carbon skeleton, and also the isonitrile group is not as closely surrounded by the methyl groups found in TMB which may block the effect ofthe active group.

EXAMPLE 8

A silicone based low surface-energy paint (RTV11) provided by General Electric Research Coφoration was used to coat fiberglass rods (10 cm length) for a one month field trial at Pearl Harbor. Pure crystalline ICP was added to the liquid paint, which hardens upon the addition of a catalyst. A 10 ppm ICP stock was prepared, mixed well, then diluted with paint for 1 ppm and 0.1 ppm concentrations. Rods were primed, then dipped into the paint after catalyst had been added. Rods coated with paint without ICP was used as a control along with plain fiberglass. The coating was allowed to cure for one week, then the rods were mounted in a random array on 22 cm² PVC pipe frames with Vexar mesh, secured by surgical rubber rings used as grommets. The frames were immersed in Pearl Harbor beneath a raft at Hospital Point. At one week intervals, 5 rods of each type were removed and the settlement of organisms counted. Percent cover of each organism was estimated using a dissecting microscope. Differences in settlement between treatments were analyzed using a Kruskal-Wallis Chi-square approximation. The results are shown in FIGURE 6A for hardfoulers, FIGURE 6B for softfoulers, FIGURE 7 for H. elegans, FIGURE 8A for diatoms, and FIGURE 8B for arborescent hydroids. The settlement of hardfoulers: calcareous tubeworms, bivalves (mostly oysters), bryozoans and barnacles and softfoulers: diatoms, arborescent hydroids, tunicates, mudtubes (polychaetes and amphipods) and sponges, are grouped in separate graphs in FIGURE 6. Silicone coated rods with 10 μg/ml ICP (ICP 10) showed significantly less settlement of all organisms for the first two weeks (p<0.01), and of diatoms on the third week. Silicone coated rods with ICP concentrations of 1 and 0.1 μg/ml also showed significantly lower settlement the first two weeks for most organisms. Epoxy paint with ICP did not have significantly lower settlement than the epoxy paint control. Settlement was significantly lower in both paint control treatments than in the fiberglass control, which is expected to show optimal settlement.

This field trial indicates that settlement of a variety of organisms is reduced under field conditions, and that ICP is apparently stable in the silicone coating used. In this trial the hardfoulers, particularly Hydroides elegans, were most strongly affected. The fact that the silicone paint control showed less fouling than the fiberglass rods is typical, and is probably related to the surface characteristic of this very slippery coating. The epoxy paint also showed less fouling than the fiberglass control, and reduction in settlement was less dramatic than with the silicone coating, with no significantly different values, although trends seem consistent. The silicone formulation is definitely superior. EXAMPLE 9

The synthetic isonitrile 1,6-diisocyanohexane (DCY) was dissolved in seawater that had been filtered through a 0.22 μ porosity filter to give a final concentration of 10 μg/ml. Dilutions were made from this stock solution for experimental trials in which the effects of concentrations between 0.05 and 10 μg/ml were tested on larvae of the tubeworm Hydroides elegans, using the bioassay procedure of Example 3. Approximately 50 competent larvae (5 days old) were added to each of five replicate dishes for each treatment. Control treatments included a positive control with a Vexar strip in seawater, which should give maximal settlement of larvae, and a negative control with only filtered seawater which should have relatively low larval settlement. Settlement was counted after 24 hours as number of tubes present, and total settlement was counted at 5 days, at which time metamorphosis was high in the control treatments. Larvae were fed phytoplankton on the second day. Settlement was expressed as a proportion ofthe total in each dish.

Four separate trials were completed using treatments of 0.1, 1 and 10 μg/ml DCY. The results are shown in Table 15:

Table 15 Mean Proportion of Hydroides elegans Larvae Settled

Mean ± SD

Treatment _Day_ Trial 1 Trial 2 Trial 3

Filtered seawater 1 0.03 ± 0.03 0.04 ± 0.05 0.04 ± 0.05

Vexar 1 0.77 ± 0.16 0.74 ± 0.18 0.65 ± 0.18

0.1 μg/ml DCY 1 0.91 ± 0.09 0.68 ± 0.29 0.59 ± 0.10

1 μg/ml DCY 1 0.90 ± 0.06 0.50 ± 0.29 0.61 ± 0.27

10 μg/ml DCY 1 0.44 ± 0.24 0.35 ± 0.26 0.21 ± 0.09

Filtered seawater 5 0.12 ± 0.09 0.96 ± 0.06 0.49 ± 0.09

Vexar 5 0.99 ± 0.02 1.00 ± 0.00 0.97 ± 0.04

0.1 μg/ml DCY 5 0.94 ± 0.04 1.00 ± 0.00 0.97 ± 0.04

1 μg/ml DCY 5 0.94 ± 0.04 0.98 ± 0.04 0.89 ± 0.06

10 μg/ml DCY 5 0.45 ± 0.23 0.68 ± 0.21 0.23 ± 0.13

The foregoing procedure was repeated at DCY concentrations of 0.05, 0.5 and 5 μg/ml. The results are shown in Table 16. Table 16 Mean Proportion of Hydroides elegans Larvae Settled

Treatment )ay Mean ± SD

Filtered seawater 0.01 ± 0.01

Vexar 0.73 ± 0.09

0.05 μg/ml DCY 0.79 ± 0.10

0.5 μg/ml DCY 0.69 ± 0.15

5 μg/ml DCY 0.57 ± 0.12

Filtered seawater 5 0.02 ± 0.02

Vexar 5 0.96 ± 0.03

0.05 μg/ml DCY 5 0.91 ± 0.03

0.5 μg/ml DCY 5 0.86 ± 0.04

5 μg/ml DCY 5 0.78 ± 0.11

This experiment indicates that 5-10 μg/ml DCY significantly inhibits settlement of H. elegans larvae (p<0.05). An interesting finding emerged: after several days of exposure to higher concentrations of DCY, the tubes of the young worms became thin and apparently decalcified.

While the preferred embodiment of the invention has been illustrated and described, it will be appreciated that various changes can be made therein without departing from the spirit and scope ofthe invention.

Claims

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. A method for preventing the attachment of underwater fouling organisms on a surface comprising treating the surface with an anti-fouling composition comprising at least one isonitrile compound.

2. The method of Claim 1 wherein the isonitrile compound is isolated from natural biological material or is synthetically produced.

3. The method of Claim 2 wherein the isonitrile compound comprises at least one isonitrile group attached to a hydrocarbon moiety comprising at least six carbon atoms.

4. The method of Claim 1 wherein the isonitrile compound is 9-isocyanopupukeanane, or an analog thereof.

5. The method of Claim 1 wherein the anti-fouling composition is applied to the surface as a liquid.

6. The method of Claim 5 wherein the anti-fouling composition further comprises a film-forming ingredient.

7. The method of Claim 6 wherein the film-forming ingredient is selected from the group consisting of chlorinated rubber resin, vinyl acetate resin, acrylic resin and natural resin.

8. The method of Claim 8 wherein the anti-fouling composition comprises about 0.1-50% by weight ofthe isonitrile compound.

9. The method of Claim 6 wherein the anti-fouling composition comprises about 0.1-70% by weight ofthe isonitrile compound.

10. The method of Claim 1 wherein the anti-fouling composition is applied to the surface as an emulsion.

11. The method of Claim 10 wherein the emulsion further comprises a surfactant.

12. The method of Claim 11 wherein the emulsion comprises about 0.1-50% by weight ofthe isonitrile compound.

13. An anti-fouling coating comprising a compound having at least one antifouling isonitrile moiety and at least one film-forming coating agent.

14. The coating of Claim 13 wherein the film-forming coating agent is a polymer selected from the group consisting of polysiloxanes, substituted polysiloxanes, modified polysiloxanes and/or copolymers thereof.

15. The coating of Claim 14 wherein the film-forming coating agent is a poly(dimethyl-siloxane) polymer.

16. The coating of Claim 14 which comprises about 0.01% to about 10% of isonitrile by weight based on the weight ofthe anti-fouling coating.

17. The coating of Claim 14 wherein the isonitrile moiety is incorporated directly into the polymer chain.

18. The coating of Claim 17 wherein the film-forming coating agent is a polysiioxane having the formula:

wherein R--_^ and R₂ are independently selected from loweralkyls having from 1 to 10 carbon atoms, e.g., methyl, ethyl, propyl, butyl, pentyl, etc.; aryl, e.g., phenyl, tolyl, xylyl, etc.; cycloalkyl, e.g., cyclohexyl, cycloheptyl, etc.; or aralkyl, e.g., benzyl, phenylethyl, etc.; either alone or substituted with one or more isonitrile groups; and X and Y represent relative percentages of the number of respective moieties in the polysiloxane, with X being less than about 10% and Y being greater than or equal to 90%) ofthe polysilane.

19. A method for preventing the attachment of underwater fouling organisms on a surface comprising treating the surface with an anti-fouling coating comprising a compound having at least one antifouling isonitrile moiety and at least one film-forming coating agent.

20. The method of Claim 19 wherein the film-forming coating agent is a polymer selected from the group consisting of polysiloxanes, substituted polysiloxanes, modified polysiloxanes and/or copolymers thereof.

21. The method of Claim 20 wherein the film-forming coating agent is a poly(dimethyl-siloxane) polymer.

22. The method of Claim 20 wherein the isonitrile moiety is incorporated directly into the polymer chain.

23. The method of Claim 23 wherein the film-forming coating agent is a polysiioxane having the formula:

wherein R_j and R₂ are independently selected from loweralkyls having from 1 to 10 carbon atoms, e.g., methyl, ethyl, propyl, butyl, pentyl, etc.; aryl, e.g., phenyl, tolyl, xylyl, etc.; cycloalkyl, e.g., cyclohexyl, cycloheptyl, etc.; or aralkyl, e.g., benzyl, phenylethyl, etc.; either alone or substituted with one or more isonitrile groups; and X and Y represent relative percentages of the number of respective moieties in the polysiloxane, with X being less than about 10% and Y being greater than or equal to 90%) ofthe polysilane.