Novel, Ecologically Safe and Economically Viable Natural Alternatives to Tributyl Tin
Proceedings of AddCoat 2001 Conference Paint Research Association
The oceans still represent our ‘last frontier’ on the earth. In spite of the decades of research on corals and deep oceans, more than 95% of the ocean remains unexplored. Marine scientific research has demonstrated to us how fragile the ocean ecosystems can be and how readily the chemicals (pesticides, industrial wastes, etc.) generated by the last 50 years of the ‘chemical revolution’ threaten human health and earth biodiversity. Among these persistent organic chemical pollutants is tributyl tin (TBT), a toxic heavy metal incorporated in marine paints that prevent attachment of fouling organisms (algae, oysters, mussels, barnacles, tube worms) to the submerged portion of the ship hull. Biofouling is a concern for the shipping industry since it impedes the movement of ships and enhance corrosion of the hull.
Over the centuries, solutions to the fouling problem have evolved from the very primitive to the very sophisticated. The earliest recorded attempts by mariners to prevent fouling dates back to the 5th century B.C. During the following centuries, the main form of protection for wooden ships was copper sheathing or the use of a mixture containing sulphur and arsenic. It was not until the development of iron hulls was copper sheathing abandoned because of serious galvanic problems. In 1625 William Beale was the first to file a patent for a paint composition containing iron powder, copper and cement. Further advancements in antifouling paints did not come about until after the Second World War with the advent of the chemical revolution that produced a wide range of industrial chemicals for new industries. A major breakthrough in antifouling technology occurred in the 1970’s when paint companies began to use TBT as a toxicant. The development of self-polishing paints that permit continuous release of TBT has revolutionized the ship coatings industry and permitted longer interval of dry-docking.
Unbeknown to its original designers however, long-term performance of TBT impregnated paints carried with it a heavy environmental price. By the 1980’s and 1990’s, TBT has been blamed for environmental damage in marine ecosystems to such an extent that the International Maritime Organization (IMO) of the United Nations has now passed a Resolution that bans the application of TBT-containing paints by the year 2001 and a total ban on the presence of TBT on all ships by the year 2008. The IMO ban is not an isolated case but a signal of the higher level of environmental consciousness spreading on a worldwide scale and brought about by the tremendous impact that organic chemical pollutants have on our ecosystems. The environmental damage as a result of chemical pollution to human health and the ecosystem can be seen worldwide, from the pollution of our drinking water by pesticides to pollution in the marine environment by industrial chemicals, including TBT. Aggressive environmental activism and increased regulatory restrictions represent attempts to make up for the years of lax government regulations and industry apathy. The IMO Resolution and the Biocidal Products Directive of the European Union (EU) are recent examples of such regulatory control. Continuing in this direction, in December 2000, 122 countries signed an international treaty that banned 12 of the most toxic persistent organic pollutants. This is a harbinger of hard times to come for the existing biocides industry, as more new compounds will likely be added to the list in the future. Companies willing to take a long term view will have to re-think how their R&D funds will be allocated and focus on identifying new biocides that will survive the more difficult regulatory environment of the future.
The initial reaction of the coatings industry to the environmental problems associated with TBT had been to develop TBT-free antifoulings. Although this has led to innovations in formulation, such changes have only taken cared of the near-term needs of the industry. In place of TBT, many companies have optimized their coatings formulation either by increasing the copper content of their paints and/or by inclusion of biocides to augment the reduced efficiency of cuprous oxide alone. With these new developments also came increases in cost, often to two or three times that of conventional TBT-based coatings. Zinc- and copper-based biocides are non-specific and retain toxic effects and will eventually come under regulatory pressures in the years to come. In the case of silicone-based technologies, high price alone is not a deterrent to effective market entry. Such non-stick coating technologies also use surfactants and oils, which are also toxic to marine life and hence likely targets for regulations in the future.
Cuprous oxide is both a toxicant and an essential component of coatings. Though necessary in trace amounts for normal cellular function, toxicity occurs when cells are overloaded with copper since there is no existing mechanism for cells to actively remove this heavy metal from their cytoplasm. In response to the elevated copper load in marinas, the recent working conference sponsored by the University of California Sea Grant in September 2000 had recommended copper reduction programs in marine boat basins in San Diego and throughout California and advocated the promotion of alternative options to copper in antifouling paints . This conference, well attended by legislators, academia, industry and environmentalists, is indicative of the trend in pro-environment stance that will eventually lead to the eventual ban on copper in the years to come in the same way that TBT has now been banned. While it has taken over a decade to ban TBT, it is likely that regulatory control will move at a faster pace given the pro-environment agenda worldwide.
The adoption of the Precautionary Principle as a guide to determine legislation against the continued use of a chemical entity presents a new paradigm. This principle states that action should be taken against a chemical when there is sufficient evidence to prove that it threatens human and animal health, even in the absence of conclusive evidence. Therefore, the search for new compounds to replace TBT and other biocides has a much stricter requirement for safety than ever before. The high cost of the discovery program and the millions of dollars needed to register new chemical entities have already deterred most companies from pursuing alternative strategies.
The search for antifouling compounds from marine organisms began in earnest in the 1970’s and continued through the 1990’s based on the observation that certain marine plants and animals naturally resist fouling. Typical examples of these are seaweeds and soft corals, notably marine sponges. Barnacles and bryozoans do not attach to these sessile organisms while they are alive. Fouling occurs only when these organisms die and presumably no longer produce the chemical defenses. This initially suggested the presence of factors produced while the animals were alive since there was no other physical mechanism for these animals to rid themselves of attached foulers. Extensive compilation of the scientific literature on all the antifouling compounds discovered to date can be found in several review articles on the subject [2,3,4].
Though the research effort has produced numerous effective antifouling agents, no single chemical has yet entered into any commercial stage. These natural substances are complex in structure and the excessive cost of synthetic production make them economically non-viable as antifouling agents. Examples of such natural compounds include renillafoulins and juncellins, which were discovered as natural antifoulants in soft corals. The cost of manufacturing such complex structures would be exorbitant. Moreover, as new chemical entities, they will have to go through the same costly regulatory process as new synthetic compounds. In addition, since large and complex molecules have multiple functions in organisms, such compounds may likely have other modes of actions that will contribute to an unsatisfactory safety profile. It is also likely that only a portion of the molecule functions as an inhibitor of attachment so that focusing on identifying the functional group of the molecules related to antifouling appears to be a more prudent use of resources.
Poseidon’s discovery program to understand the nature of the functional groups that contribute to antifouling effects in these complex molecules began in the mid-1980’s. By the mid-1990’s we began to unravel the specific structural conformations that appeared to be preferentially associated with such effects. Rather than pursuing the classic approach of using structure-activity relationships to produce chemical entities directed against barnacle settlement, we have pursued a different approach, i.e., using our understanding of the structural requirements and matching known natural products—terrestrial and marine–that comes close to such structures. This new directive, the Natural Bioproducts (NB) Screening Program, enabled us to select a wide range of biological products that mimic the target structural requirement.
The key to the success of the NB Screening Program is the ability to efficiently screen the biological effects of candidate substances. The barnacle, Balanus amphitrite Darwin, is a predominant biofouling organism found in practically all harbors in the world. It is an ideal test organism because of a once-a-week reproductive cycle, rapid larval growth, standard methods to rear them in mass cultures and a predictable settlement behavior. Upon reaching stage IV-nauplius, the larvae undergo transformation into cyprids, the non-feeding settlement stage. The cyprids ‘walk’ over a surface using antennules, which possess numerous sense organs and are able to secrete a temporary adhesive. Once a suitable surface is selected, the cyprid fixes itself on the surface by producing cement through its sucking disc. Twenty-four hours later, the cyprid moults into a pinhead barnacle and becomes firmly entrenched on the surface. The process of attachment is a complicated event, with a myriad of environmental cues affecting successful settlement. The barnacle settlement assay developed by Rittschof et al  enabled Poseidon to pursue a rational approach to the selection of a series of naturally occurring compounds that we refer to as the NB Class.
Poseidon’s NB compounds were selected based on the following criteria:
• Inhibition of settlement below 0.025 mg/ml
Only a few select compounds were found to satisfy the above criteria. Compounds that were effective but expensive to manufacture were automatically eliminated for further studies. The best in the series, for proprietary reasons, is referred to as NB-17 and is profiled here briefly as an example of the characteristics of compounds discovered through Poseidon’s Natural Bioproducts Screening Program.
When subjected to the barnacle settlement assay, NB17 showed a better efficacy than juncellin, the active antifoulant found in Juncella juncea . A comparison of NB17 with other purified extracts of sponges  from the Gulf of Mannar (South India) is shown in Fig. 1. It is very encouraging for us that NB17, a mimic of the functional group in natural antifoulants, has a good efficacy profile. The EC50 for NB17 is 0.004 mg/ml, which exceeded the standard requirement of an EC50 of 0.025 mg/ml established by the US Navy program as an efficacy level for natural antifoulants.
To test the performance of NB17 under field conditions, iron panels were painted with a conventional paint composition containing cuprous oxide as toxicant in the presence of 2% NB17. The experimental and control panels without NB17 were continuously submerged in San Dionisio Bay (Philippines) for a period of 1.5 years. This protected bay area is rich in planktons and in biofouling organisms. The primary foulers are Balanus amphitrite communis and the rock oyster, Crassostrea cuculata. Typical examples of fouling after 1.5 years are shown in Fig. 2. Hard fouling became evident in the control panels by the 9th month and after one year; the control panels (without NB17) were totally encrusted with barnacles. In comparison, the experimental panels were practically devoid of fouling, except in minute areas where there was physical damage to the painted surface.
Besides efficacy, toxicity is a major issue of concern. Even natural compounds can be as toxic as heavy metals. NB17 will need to be safe enough to meet the guidelines of the Precautionary Principle even before more extensive testing can be justified.
We have also examined the toxicity profile of NB17. The data shown in Table I are from a typical acute toxicity study on the effect of exposure of marine larvae to NB17. The lethal dose to barnacle larvae (LD50) for NB17 is 0.6 mg/ml. In the case of the nauplii of the brine shrimp, Artemia salina, as test organism, we have found that the LD50 was 0.75 mg/ml. The LD50 for TBT is approximately 0.0000000034 mg/ml. Thus, we are observing a considerable margin of safety in the use of NB17 as an antifoulant considering that the effective concentration was 0.004 for NB17.
Additional data (not shown here) also indicated that NB17 has antibacterial activity. In a panel of marine bacteria normally associated with barnacles, we found effective inhibition against Flavobacterium sp and Aeromonas sp. Other bacterial strains were moderately sensitive. NB17 has also been shown to be inhibitory to marine microalgae such as Dunaliella tertiolecta and Nitzchia sp.
Rittschof  has pointed out that the industry will have to look at its antifouling R&D program with a different point of reference. If the point of comparison for efficacy uses existing potent biocides and toxicants as reference, then natural antifoulants with very low toxicity profiles cannot be expected to meet industry expectations. Natural antifoulants that are non-toxic are likely to work either through a repellent mechanism by disrupting specific cellular functions associated with attachment or as minor irritants rendering the surface unfavorable for attachment, parameters untested or unused by existing toxicants. The processes involved in the attachment to surfaces by diverse organisms are likely to be also varying from one class of fouling organism to another. A single natural antifoulant, on its own, will probably not meet industry requirements. We also envisage that a combination of antifoulants working through different mechanisms will likely provide the spectrum of responses that can meet the needs of the industry.
Poseidon has chosen NB17 as its primary candidate because of the efficacy and safety noted above. The cost of manufacturing this compound is also quite low and comparable to the raw material costs for toxicants like TBT. There are many other aspects that need to be addressed such as compatibility with various paint formulations, leaching rate and long-term environmental impact. In spite of the hurdles ahead, we are encouraged by the performance of NB17 as a potential novel antifouling agent.
1. Alternative antifouling strategies for recreational boats: a working conference. University of California Sea Grant program, US Department of Commerce & Agriculture and County for San Diego. September 21-22, San Diego Yacht Club, San Diego, California.
2. Rittschof, D. Natural product antifoulants: one perspective on the challenges related to coatings development. Biofouling, 10, 1, 2000.
3. Davis AR, Targett NM, McConnell OJ, Young CM. Epibiosis of marine algae and benthic invertebrates: Natural products chemistry and the mechanisms in inhibiting settlement and overgrowth. In Marine Organic Chemistry, vol. 3, Scheuer PJ, ed, Springer Verlag, Berlin, 1989, 85.
4. Clare, AS. Marine natural products antifoulants: status and potential. Biofouling, 9, 211, 1996.
5. Rittschof D, Hooper IR, Branscomb ES, Costlow JD. Inhibition of barnacle settlement and behavior by natural products from whip corals, Leptogorgia virgulata (Lamarck, 1815). J Chem Ecol, 11(5), 551, 1985.
6. Mary V, Mary A, Rittschof D, Sarojini R, Nagabushanam R. Compounds from octocorals that inhibit barnacle settlement: isolation and biological potency. in Bioactive Compounds from Marine Organisms, Thompson MF, Sarojini R, Nagabushanam R, eds. Oxford & IBH Publications, New Delhi, 330, 1991.
7. Mary A, Mary V. Barnacle antifouling natural products from marine biota. in Barnacles: The Biofoulers, Thompson MF, Nagabushanam R, eds., Regency Publications, New Delhi, 379, 1999.