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Developing new marine antifouling substances: learning from the pharmaceutical industry
11 Developing new marine antifouling substances: learning from the pharmaceutical industry L MÅRTENSSON LINDBLAD, University of Gothenburg, Sweden
Abstract: The search for the ideal antifouling substance has a long history. In times before occupational health and environmental concerns, efficacy was the only parameter that was taken into account. However, that has dramatically changed during the last thirty years. It is now necessary to understand the regulatory framework of the area to ensure safety, and combine that with an understanding of the economic realities that in the end have to pay for the new developments. A sector that has successfully implemented efficacy, safety and market economy is the pharmaceutical industry. Scientific efforts, industrial development and regulatory framework have together created novel science as well as new and safer products. Today, the search for new antifouling substances shares many of the features experienced by the pharmaceutical industry. For example, scientific knowledge in biology, development of a control release system, production costs and how to prove the product safe for the end consumer, independent of man or nature. Key words: product development, regulatory framework, antifouling substances, pharmaceuticals, bioassays, neurotransmitter receptors.
11.1
Introduction
As long as mankind has used the sea for transportation, marine biofouling has been an issue. When the seafarers were few and the ocean infinite and when there was no occupational health care, the biological growth on a ship hull could be avoided by using such toxic compounds as lead, mercury or arsenic. Compounds with high general toxicity could kill off all adhered organisms, regardless of whether they were algae, bacteria or invertebrates. With the increased awareness that non-target organisms as well as humans can be affected by antifouling substances, new regulatory demands have been introduced as well as an increased scientific effort to find new substances that will satisfy new demands. The strategy in how to find those substances was phrased 50 years ago. 263
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While interest in the biological aspects of fouling may appear to end with the discovery of toxic coatings capable of preventing the growth, it should be remembered that new protective devices can not very well be developed without a fundamental understanding of the fouling populations. Marine Fouling and Its Prevention. Contribution No 580 from Woods Hole Oceanographic Institute 1952.
However, toxic coatings, especially regarding the tributyltin (TBT) era, were not without complications, of which we are now well aware. But do we have new solutions today or are we re-using the same knowledge in a different way? The first intuitive answer is that yes, of course we have come up with new solutions, but the second thought is, no, we are still using copper and to some extent, less favourable biocides on most ship hulls or other marine constructions. A huge effort is now available resulting in a vast range of modern coatings with much higher performance and efficiency, but the biology part of the two-side problem is dragging behind. Why so? One may speculate. A contributing factor may be that biofouling is a truly multidisciplinary research and as such, it is not suitable in the academic tradition and outside the scope of the coating industry. However, the fact that biofouling cannot be solved without different area of competence was also recognized in 1952: Fouling is, however, a biological phenomenon. If it is to be dealt with effectively from an engineering point of view, it is important that the biological principles which determine its development be understood.
In the world of today, if one addresses biofouling in a structural way, competences are needed not only in general marine biology or paint formulation, but in subdisciplines such as ecotoxicology, molecular biology and surface chemistry, but even more important an understanding of regulatory science. A contributing triad has to be constructed (Fig. 11.1).
11.2
The communication triad
The triad is built on three main players and three different components. Most of the regulatory framework is set by political decisions to protect citizens and the environment, with authorities to implement those decisions. Paint formulation skills and knowledge is mostly associated with paint companies, and more explorative research is performed by different academic groups. The major difference from the 1952 report is development within environmental sciences and as a result thereof, the upcoming of regulatory science seen as new regulations and legislations and the implementation thereof. The triad is rather new within the area of marine biofouling, but as
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Regulatory bodies
Communication Academia
Producers
11.1 The communication triad. Three main players are involved in the development of new antifouling substances. The regulatory body has as its purpose the protection of man and the environment. Their demands are directed towards the producers that need to fulfil the regulatory demand of information. A third player, the concept providers, academia, are traditionally less aware of market and regulatory demands, thereby reducing the ability to choose the right strategy and substances for product development. With a more interactive discussion between different components of the triad, a better innovation climate can be created.
such, it is a well-developed model in other areas such as the pharmaceutical industry. Paint formulation has a contra part in pharmaceutical formulation, since the task is the same: controlling release of potent chemicals over time. Another similarity between the two areas seems to be an incidence of harmful side effects as a wake up and starting point in developing regulatory science. The pharmaceutical industry had its thalimodide (Neurosedyne) incident in the late 50s and regarding antifoulants, TBT in the 80s. A defined goal for all parts of the triad is to develop new products, with less risk, with effective substances that can be incorporated into a final product that has market compliance. We all want to find solutions for the market that are in every respect better than the older ones: safer for the environment and with higher efficacy, giving better performance. The different positions in the communication triad are all equally necessary for developmental work but it is also necessary to understand the different roles. Since it is a communication triad, for success it points towards the fact that within such a multidiscipline area as antifouling, I as a scientist have to orientate myself toward understanding at least parts of the regulatory framework as well as market demands regarding products and relate that back to the latest developments within science. As we stand now, the market does not have unlimited resources nor does science, while society demands increasing assurance of safe, and more
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effective, products. Neither the paint industry nor academia are capable of finding resources demanded by regulatory bodies for new substances. A new product as it is now, is associated with risks higher than the market may be willing to take or pay. To be able to push the area forward, e.g. coming up with new substances, it is vital that all parts are willing to agree on risks and share the burden of risk assessment.
11.3
From concepts to products
Proof of mechanism
Risk assessment
Control release
Biology
Toxicology
Formulation
New chemical entity
Regulatory efforts
Proof of concept
Market analysis and introduction
Many concepts are not fruitful, lacking components for successful market introduction or perhaps even an insight that a substance (concept) is not immediately a product. As a multidisciplinary research area, a developmental model is composed of different interactive boxes with different expertise (Fig. 11.2). At certain times, one speciality may be of more importance but none can be missing. As the demands are increasing in all aspects, a more structural analysis of resources and competences needs to be performed in projects regarding biofouling control. A key issue in the developmental work is how to transfer concepts to market acceptable products. Scientists are often the concept providers, and the paint (or chemical) industry the product producers and market providers. Proof of concept is the bridge
11.2 A development model. All horizontal boxes represent concepts and the critical process when concepts must be transferred into products. Within that process, horizontal boxes must relate to the vertical boxes complying with regulatory and market demands in an interactive way. That process is best described as ‘proof of concept’ and it is critical in the development process. A hindrance may be different opinions in what is needed and what has to be proven. That may hamper, or in the worst case, put an end to the process.
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between the two. It should contain field efficacy and a basic set of toxicological testing, preferably at least degradation, bioaccumulation, cell toxicity and assurance that the suggested substance does not induce mutagenecity or bind to DNA-molecules. From my point of view, being a concept provider also includes assurances that the new substance may be suitable as a new antifoulant, and also have no mutagenic or bioaccumulative properties. Proof of concept is the most critical step associated with high risks in the innovation process and is not regularly discussed, and in some cases is referred to ‘not being my problem’. A functional communication triad is necessary to overcome problems associated with proofs of concept; to build collaboration linking concepts into products.
11.3.1 There is no single solution The biological complexity of the phenomenon we call biofouling is enormous. It is an ecological community with entities originating from all that we call life. Also, each organism has its own solution for how to find and stay firm on a surface, evolved during millions of years. In my view, it is impossible to invent new antifouling substances without restricting the problem, meaning that new molecules have to be part of a bigger solution. Any new substance will have an efficacy profile that differs from barnacles compared to algae (Konstantinou and Albanis, 2004). Therefore, it seems to be more logical and rational to use a combination of different substances to reduce the overall usage of marine biocides. By being more precise in mode of action, identifying key biological components in the biology of surface attachment, it is possible to reduce the amount of the biocide.
11.3.2 Rational development needs multidisciplinary action: the barnacle example As all organisms have their own solutions in adhering, we may also find several new ways to combat marine fouling and it is likely to be driven forward by increased knowledge in relevant sciences. There is a need to understand the general fouling biology, an increased knowledge in the area between traditional marine biology and surface chemistry. In our experience, based on cyprid larvae of the species Balanus improvisus, it is not difficult to demonstrate antifouling effects in laboratory of most substances in concentrations about 1 μM and above (Rittschof et al., 2003; Dobretsov and Qian, 2003; Dahlström et al., 2005). Without confirming a mode of action, this is not satisfactory from a scientific point of view. It points towards the fact that to understand attachment mechanisms, it is not enough to perform experiments with pharmacological molecules, without knowing if the biological targets exist in barnacles or other fouling organisms. It has
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to be combined with a number of different assays and experimental approaches (see Chapter 12: Dahms and Hellio).
11.4
Learning from the pharmaceutical industry
A focus within the pharmaceutical industry has always been to know the physiology as well as pathology within a chosen area. To be able to develop a pharmaceutical against high blood pressure, it is essential to identify hormones and neurotransmitters that contribute to blood pressure regulation and their specific receptors. Especially the receptors have proved to be fruitful targets in the development of small molecules that could alter the physiological output in a more favourable way. When a receptor, or ion channel or enzyme, has been identified as a key target for pharmaceutical treatment, it is then possible to screen for new substances in a systematic way. With molecular techniques, it is possible to isolate and develop biological assay with the target protein, a receptor of a specific hormone or neurotransmitter. With the isolated protein, expressed in a bioassay, the chemists will be able to create a vast number of derivate molecules which become a chemical library that can be tested against the target protein within an appropriate bioassay. In such a way, it is possible to create a high throughput system that combines knowledge and serendipity. The development that has made this possible is most of all knowledge in basic physiology and the ability to adopt modern technology within test systems that could become new bioassays. This methodology is also a possibility within antifouling research. An objection is that there are too many and too different types of organisms regarding marine biofouling so that it would be impossible to introduce such systematic research routes. However, the most basal physiological systems are similar between species. It is possible to identify targets and perform high throughput systematic research using general systems such as neurotransmitter receptors. Barnacles are no different from most other organisms. They do have neurotransmitters such as dopamine (Okano et al., 1996), serotonin (Yamamoto et al., 1996; Yamamoto et al., 1999), histamine (Callaway and Stuart, 1998), acetylcholine (Faimali et al., 2003) and probably also octopamine. Barnacle octopamine receptors have been identified and the receptor is known from other crustaceans. However, usually noradrenaline or adrenaline are not thought to act as neurotransmitters within invertebrates but are exclusive for vertebrate species (Roeder, 2005). There are some indications that noradrenaline may affect settling and metamorphosis (Yamamoto et al., 1996; Okano et al., 1996) in higher concentrations, further supported by antisettling effects seen with phentolamine (Yamamoto et al., 1998). However, phentolamine and yohimbine are also
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known as octopamine antagonists (Evans and Robb, 1993; Roeder, 2005; Maqueira and Chatwin, 2005) and noradrenaline binds as well towards dopamine receptors (Degen et al., 2000). Any results suggesting receptor specificity using the settling assay must be regarded with some care. The term specificity is a relative term based on mammalian pharmacology where the minutiae differences between receptor subtypes have been studied. Claiming specificity needs comparative studies between different receptor types. There are few such studies regarding invertebrate receptors and none within the area of antifouling. Instead, several adrenergic compounds bind to octopamine receptors (Evans and Robb, 1993; Howell and Evans, 1998) and dopamine receptor pharmacology within invertebrates is different compared to what is known from vertebrate, especially mammalian, studies. (Degen et al., 2000; Ödling et al., 2007; Zega et al., 2007). Any claim of specificity must be carefully evaluated on its own merits. What is lacking is knowledge regarding receptor biology and pharmacology, especially when looking at future rational development within the area. Only two receptors have been cloned (Isoai et al., 1996; Kawahara et al., 1997), both octopamine receptors (GPR18_BALAM (Q93127) and GPR9_BALAM (Q93126); www.expasy.org). A successful pharmacological approach must include detailed knowledge regarding receptor biology in terms of affinity, efficacy and specificity. This does not mean that we need to characterize every species. There are similarities between species even if science tends to emphasize the differences. As seen above, dopamine is a ubiquitous neurotransmitter and substances that have been developed for human diseases do affect barnacle cement secretion (Ödling et al., 2006). But to ensure that the right receptor protein is targeted, more biological assays need to be developed as well as molecular biology tools need to be applied in future developmental work. Regarding medetomidine as an example of a new antifoulant, the goal has been to find a reversible mechanism that does not necessary kill the cyprid larva, only prevent settling. We know that medetomidine binds to a receptor; the effect is reversible and specific antagonists have been identified (Dahlström et al., 2000; 2005). The mode of action is activating swimming movements that will short cut the surface behaviour (Hasselberg Frank, in manuscript). At the same time, the surface is nevertheless of great importance. Medetomidine is a surface active molecule (Dahlström et al., 2000; 2004). The receptor responsible for medetomidine activity within barnacles has now been cloned and been transferred into a yeast and been expressed within yeast cells (see Lind; 14th ICMCF conference, Kobe, Japan). This opens up the possibility to find and explore future substances that can be found within a chemical library that has the right biological and chemical properties that could either activate or block the receptor. In similar way,
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any identified receptor, for example a dopamine receptor, can be transferred over into an expression system and become the target in evaluating a chemical library designed for invertebrate dopaminergic receptors. The differences between pharmaceutical and antifouling development is the life cycle analysis of the biological active substance. To be included within a paint matrix or absorbed in the gastrointestinal system are two different means of distribution. A paint system may want a more lipophilic substance, whereas a more hydrophilic may be advantageous regarding a pharmaceutical. Degradability is a key issue within antifouling to avoid bioaccumulation, whereas a high degradation rate might not be favourable regarding pharmaceuticals, since it might mean a more complicated dosing regimen. If the biological target is identified and high throughput screening methodology implemented, it is possible to find substances that could meet biological efficacy criteria as well as having acceptable chemical properties. However, the ideal antifouling (or pharmaceutical substance) will never be found. There will always be compromises between the biological, chemical and environmental properties to decide between. Whether those decisions are right or wrong, that is the task for the regulatory system.
11.5
The importance of formulations
The paint industry has for centuries provided the market with different products and solutions. Most of the knowledge is empirical. Paint formulation is in many respects a difficult task, since small amounts of a new ingredient may totally change the physical and chemical properties. Rationally, it would be easier if incorporated biocides were a minor part of the formulation. At the same time, no one can foresee how mixtures may interact with each other and the formulation per se. The paint formulation is at the same time a storage and release system for the antifouling substance. As such, it plays a key role regarding efficacy and performance. In laboratory tests, medetomidine and a sister compound, clonidine are almost equally effective (Dahlström et al., 2000), but in field they differ totally. While medetomidine will stay completely effective for over two years, clonidine does not last for a single season on the Swedish west coast (unpublished data). This difference in efficacy is likely to be associated with surface behaviour of the molecules. Leakage of a new substance is one key issue when assessing environmental risks. Models like MAMPEC (free download from www.cepe.org) used to predict Predicted Environmental Concentration (PEC) is balancing between degradability, toxicity and leakage. A substance with low leakage rate is less sensitive to factors such as non-target toxicity or degradability. Leakage is also a parameter that can be refined by providing a control release system. A new tendency within paint formulation is to use
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microcapsule techniques also known from the pharmaceutical industry (Zhang et al., 2007).
11.6
Side effects and regulation
Dealing with such complex biological mechanisms, claiming efficacy and not expecting to find any side effect is a bit naïve. The question is more whether the side effects are acceptable and what the risks are with a specific substance. We may all conclude that every new antifouling substance will be hazardous, but the question we ought to answer is whether the risk is acceptable. In those terms, it is a great advantage if the mode of action is known. Is it a common biological mechanism that is a target and does it act through toxicity alone? Then the degradation data will become more critical, since general mechanisms are generally more hazardous. This is seen with substances such as Sea-Nine which has a high degree of toxicity but is quickly degradable and not bioaccumulative (Willingham and Jacobson, 1996; Jacobson and Willingham, 2000). The risks are therefore acceptable. Medetomidine is an example of the opposite way of dealing with side effects. It is specific and is fully reversible (Dahlström et al., 2000). Tests can be set up to predict foreseeable side effects from what is already known regarding its mode of action. The hazard is less, but still risks need to be quantified. Even though medetomidine is not hydrolyzed and degraded as quickly as Sea-Nine 211, it is not bioaccumulative (Hilvarsson, 2007) and its release rate is in ng/cm2/day (Dahlström, 2004; Borgert, 2004). Those features allow medetomidine to be used as an antifouling substance.
11.6.1 The regulatory process A way of handling risks from the society perspective is to have a regulatory procedure. Regarding Europe, this is now being transformed from the national level towards a European legislation. This is summarized in the Biocidal Product Directive (BPD) 98/8. For many European countries it changes the legislation regarding antifouling products. In most European countries notification of antifouling substances has been necessary but not a regulatory process. These issues are discussed in Chapter 10.
11.7
Marketing a new product
No development efforts are without a price, and antifouling development must in the end be economically sound. The product per se must be priced such that it may enter the market – but still not be underfinanced, since that will stop any further development. No one will ever say:
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We are prepared to take the costs for a new antifoulant.
In a broader perspective, this means that for a successful and a more dynamic market, we as scientists must learn more about issues beyond science if we are to be realistic in our hopes for our results and inventions. We must learn that to make a compound a success, it means more than efficacy in lab and field; it also means dealing with and judging the outcome from much more difficult aspects such as formulation, toxicological risks, as well as keeping on an eye on the market. In that process we need to interact with other experts, regulatory authorities as well as industrial competences. Once again, probably the most successful industry in terms of profit, the pharmaceutical sector, learned this lesson much earlier than the biocide sector. The sooner we create a more dynamic viewpoint, the more successfully will science come up with new possible and realistic alternatives that could interact with authorities and industry. As part of a three communication triad, industry and authorities need to have the ability and willingness to interact with new dynamic approaches and possibilities within science. When comparing the antifouling sciences with the pharmaceutical industry, one must also be aware of the differences between those industry sectors. Most of all, the pharmaceutical industry market is, in terms of money, much bigger than the antifouling paint market. It means that the driving forces and the risks that venture capitalists are willing to take are also higher. Therefore, pharmaceuticals are subsidized in many societies and therefore not comparable with the antifoulant market. It means that the innovation rate within the pharmaceutical industry can be faster due both to market size as well as society contribution measured as efforts and resources invested in science. Efforts are also driven by emotional factors since pharmaceuticals concern us as human beings. This is not the case regarding antifouling, although the increased awareness of the marine environment has been raised, also seen as new regulatory standpoints. Scientifically, the pharmaceutical and antifouling development processes are similar; find a substance, formulate and register. Although the overall biological complexity within the area of marine biofouling is overwhelming, especially knowing the lack of background information that exists in biomedicine and clinical sciences, as a matured industrial sector, pharmaceutical development serves as an example to reflect upon.
11.8
Summary
In many ways, antifouling research is still a very young scientific area, although efforts to protect marine surfaces have been an issue for mankind as long as the sea has been used for transportation. Our main and most
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used antifouling substance is still copper, used as an antifouling substance in paints since the 19th century. However, with the increased awareness of the marine environment and the new regulatory situation, this might change. There is an expectation that more substances will be banned and therefore create a demand for new ones with fewer risks, developmental work including academia, industry and society. Although, the trend can be the complete opposite due to the costs associated with the regulatory demands, causing new innovations to become economically impossible and therefore damage future opportunities for a better marine environment. In the long run, it might also be a dead end for market providers in that the alternatives will become fewer, reduced to few products with many producers. This is more of a political issue, but if we are serious in wanting new environmentally friendly products, we need all parts of the communication triad to share risks and development costs. In my veiw, new products are necessary to prove success for all parts involved, regardless of being concept or market providers.
11.9
References
Borgert, T. (2004). Investigation of interaction between medetomidine and alkyd using dynamic dialysis. Gothenburg: Chalmers University of Technology. Callaway, J. & Stuart, A. (1998). The distribution of histamine and serotonin in the barnacle’s nervous system. Microscopy Research and Techniques, 44 (2–3), 94–104. Dahlström, M. (2004). Pharmacological agents targeted against barnacles as lead molecules in new antifouling technologies. Gothenburg: University of Gothenburg. Dahlström, M., Jonsson, P. R., Lausmaa, J., Arnebrant, T., Sjögren, M., Holmberg, K. et al. (2004). Impact of Polymer Surface Affinity of Novel Antifouling Agents. Biotechnology and Bioengineering, 86 (1), 1–8. Dahlström, M., Lindgren, F., Berntsson, K., Sjögren, M., Mårtensson, L., Jonsson, P. et al. (2005). Evidence for different pharmacological targets for imidazoline compounds inhibiting settlement of the barnacle Balanus improvisus. Journal of Experimental Zoology, 303A (7), 551–562. Dahlström, M., Mårtensson, L., Jonsson, P., Arnebrant, T. & Elwing, H. (2000). Surface active adrenoceptor compounds prevent the settlement of cyprid larvae of Balanus improvisus. Biofouling, 16 (2–4), 191–203. Degen, J., Geweke, M. & Roeder, T. (2000). The pharmacology of a dopamine receptor in the locust nervous tissue. European Journal of Pharmacology, 396, 59–65. Dobretsov, S. & Qian, P.-Y. (2003). Pharmacological induction of larval settlement and metamorphosis in the blue mussle Mytilus edulis L. Biofouling, 19 (1), 57–63. Evans, P. & Robb, S. (1993). Octopamine receptor subtypes and their modes of action. Neurochemical Research, 18 (8), 869–874. Faimali, M., Falugi, C., Gallus, L., Piazza, V. & Tagliafierro, G. (2003). Involvement of acetyl choline in settlement of Balanus amphitrite. Biofouling, 19 (1), 213–220. Hilvarsson (2007). The antifoulant medetomidine. Sublethal effects and bioaccumulation in marine organisms PhD. thesis. University of Gothenburg, Dept of Marine Ecology.
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Howell, K. & Evans, P. (1998). The characterization of presynaptic octopamine receptors modulating octopamine release from an identified neurone in the locust. The Journal of Experimental Biology, 201, 2053–2060. Isoai, A., Kawahara, H., Okazaki, Y.-I. & Shizuri, Y. (1996). Molecular cloning of a new member of the putative G-protein-coupled receptor gene from barnacle Balanus amphitrite. Gene, 175 (1–2), 95–100. Jacobson, A. & Willingham, G. (2000). Sea-nine antifoulant: an environmentally acceptable alternative to organotin antifoulants. The Science of the Total Environment, 258, 103–110. Kawahara, H., Isoai, A. & Shizuri, Y. (1997). Molecular cloning of a putative serotonin receptor gene from barnacle, Balanus amphitrite. Gene, 184, 245–250. Konstantinou, I. & Albanis, T. (2004). Worldwide occurence and effects of antifouling paint booster biocides in the aquatic environment: a review. Environmental Inernational, 30, 235–248. Maqueira, B. & Chatwin, H. E. (2005). Identification and characterization of a novel family of Drosophila beta-adrenergic-like octopamine G-protein coupled receptors. Journal of Neurochemistry, 94, 547–560. Ödling, K., Albertsson, C., Russell, J. T. & Mårtensson, L. G. (2006). An in vivo study of exocytosis of cement proteins from barnacle Balanus improvisus (D.) cyprid larva. The Journal of Experimental Biology, 209, 956–964. Ödling, K. (2007). The cement gland of the Balanus improvisus cyprid larva – with special emphasis on secretion and regulations. Licentiate thesis, University of Gothenburg, Dept of Zoology. Okano, K., Shimizu, K., Satuito, C. & Fusetani, N. (1996). Visualization of cement exocytosis in the cypris cement gland of the barnacle Megabalanus rosa. Journal of Experimental Biology, 199 (10), 2131–2137. Rittschof, D., Lai, C.-H., Kok, L. M. & Teo, S. L.-M. (2003). Pharmaceuticals as antifoulants: Concept and principles. Biofouling, 19 (1), 207–212. Roeder, T. (2005). Tyramine and octopamine: Ruling behaviour and metabolism. Annual Review of Entomology, 50, 447–477. Willingham, G. & Jacobson, A. (1996). Designing an environmentally safe marine antifoulant. ACS symposium series, 640, 224–233. Yamamoto, H., Satuito, C., Yamazaki, M., Natoyama, K., Tachibana, A. & Fusetani, N. (1998). Neurotransmitter blockers as antifoulants against planktonic larvae of the barnacle Balanus amphitrite and the mussel Mytilus galloprovincialis. Biofouling, 13 (1), 69–82. Yamamoto, H., Shimizu, K. & Tachibana, A. F. (1999). Roles of dopamine and serotonin in larval attachement of the barnacle, Balanus amphitrite. Journal of Experimental Zoology, 284, 746–758. Yamamoto, H., Tachibana, A., Kawaii, S., Matsumura, K. & Fusetani, N. (1996). Serotonin involvement in larval settlement of the barnacle, Balanus amphtrite. The Journal of Experimental Zoology, 275, 339–345. Zega, G., Pennati, R., Dahlström, M., Berntsson, K., Sotgia, C. & De Bernardi, F. (2007). Settlement of the barnacle Balanus improvisus: The roles of dopamine and serotonin. Italian Journal of Zoology, 74 (4), 351–361. Zhang, M., Cabane, E. & Claverie, J. (2007). Transparent antifouling coatings via nanoencapsulation of a biocide. Journal of Applied Polymer Science, 105, 3824–3833.
Abstract: The search for the ideal antifouling substance has a long history. In times before occupational health and environmental concerns, efficacy was the only parameter that was taken into account. However, that has dramatically changed during the last thirty years. It is now necessary to understand the regulatory framework of the area to ensure safety, and combine that with an understanding of the economic realities that in the end have to pay for the new developments. A sector that has successfully implemented efficacy, safety and market economy is the pharmaceutical industry. Scientific efforts, industrial development and regulatory framework have together created novel science as well as new and safer products. Today, the search for new antifouling substances shares many of the features experienced by the pharmaceutical industry. For example, scientific knowledge in biology, development of a control release system, production costs and how to prove the product safe for the end consumer, independent of man or nature. Key words: product development, regulatory framework, antifouling substances, pharmaceuticals, bioassays, neurotransmitter receptors.
11.1
Introduction
As long as mankind has used the sea for transportation, marine biofouling has been an issue. When the seafarers were few and the ocean infinite and when there was no occupational health care, the biological growth on a ship hull could be avoided by using such toxic compounds as lead, mercury or arsenic. Compounds with high general toxicity could kill off all adhered organisms, regardless of whether they were algae, bacteria or invertebrates. With the increased awareness that non-target organisms as well as humans can be affected by antifouling substances, new regulatory demands have been introduced as well as an increased scientific effort to find new substances that will satisfy new demands. The strategy in how to find those substances was phrased 50 years ago. 263
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While interest in the biological aspects of fouling may appear to end with the discovery of toxic coatings capable of preventing the growth, it should be remembered that new protective devices can not very well be developed without a fundamental understanding of the fouling populations. Marine Fouling and Its Prevention. Contribution No 580 from Woods Hole Oceanographic Institute 1952.
However, toxic coatings, especially regarding the tributyltin (TBT) era, were not without complications, of which we are now well aware. But do we have new solutions today or are we re-using the same knowledge in a different way? The first intuitive answer is that yes, of course we have come up with new solutions, but the second thought is, no, we are still using copper and to some extent, less favourable biocides on most ship hulls or other marine constructions. A huge effort is now available resulting in a vast range of modern coatings with much higher performance and efficiency, but the biology part of the two-side problem is dragging behind. Why so? One may speculate. A contributing factor may be that biofouling is a truly multidisciplinary research and as such, it is not suitable in the academic tradition and outside the scope of the coating industry. However, the fact that biofouling cannot be solved without different area of competence was also recognized in 1952: Fouling is, however, a biological phenomenon. If it is to be dealt with effectively from an engineering point of view, it is important that the biological principles which determine its development be understood.
In the world of today, if one addresses biofouling in a structural way, competences are needed not only in general marine biology or paint formulation, but in subdisciplines such as ecotoxicology, molecular biology and surface chemistry, but even more important an understanding of regulatory science. A contributing triad has to be constructed (Fig. 11.1).
11.2
The communication triad
The triad is built on three main players and three different components. Most of the regulatory framework is set by political decisions to protect citizens and the environment, with authorities to implement those decisions. Paint formulation skills and knowledge is mostly associated with paint companies, and more explorative research is performed by different academic groups. The major difference from the 1952 report is development within environmental sciences and as a result thereof, the upcoming of regulatory science seen as new regulations and legislations and the implementation thereof. The triad is rather new within the area of marine biofouling, but as
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Regulatory bodies
Communication Academia
Producers
11.1 The communication triad. Three main players are involved in the development of new antifouling substances. The regulatory body has as its purpose the protection of man and the environment. Their demands are directed towards the producers that need to fulfil the regulatory demand of information. A third player, the concept providers, academia, are traditionally less aware of market and regulatory demands, thereby reducing the ability to choose the right strategy and substances for product development. With a more interactive discussion between different components of the triad, a better innovation climate can be created.
such, it is a well-developed model in other areas such as the pharmaceutical industry. Paint formulation has a contra part in pharmaceutical formulation, since the task is the same: controlling release of potent chemicals over time. Another similarity between the two areas seems to be an incidence of harmful side effects as a wake up and starting point in developing regulatory science. The pharmaceutical industry had its thalimodide (Neurosedyne) incident in the late 50s and regarding antifoulants, TBT in the 80s. A defined goal for all parts of the triad is to develop new products, with less risk, with effective substances that can be incorporated into a final product that has market compliance. We all want to find solutions for the market that are in every respect better than the older ones: safer for the environment and with higher efficacy, giving better performance. The different positions in the communication triad are all equally necessary for developmental work but it is also necessary to understand the different roles. Since it is a communication triad, for success it points towards the fact that within such a multidiscipline area as antifouling, I as a scientist have to orientate myself toward understanding at least parts of the regulatory framework as well as market demands regarding products and relate that back to the latest developments within science. As we stand now, the market does not have unlimited resources nor does science, while society demands increasing assurance of safe, and more
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effective, products. Neither the paint industry nor academia are capable of finding resources demanded by regulatory bodies for new substances. A new product as it is now, is associated with risks higher than the market may be willing to take or pay. To be able to push the area forward, e.g. coming up with new substances, it is vital that all parts are willing to agree on risks and share the burden of risk assessment.
11.3
From concepts to products
Proof of mechanism
Risk assessment
Control release
Biology
Toxicology
Formulation
New chemical entity
Regulatory efforts
Proof of concept
Market analysis and introduction
Many concepts are not fruitful, lacking components for successful market introduction or perhaps even an insight that a substance (concept) is not immediately a product. As a multidisciplinary research area, a developmental model is composed of different interactive boxes with different expertise (Fig. 11.2). At certain times, one speciality may be of more importance but none can be missing. As the demands are increasing in all aspects, a more structural analysis of resources and competences needs to be performed in projects regarding biofouling control. A key issue in the developmental work is how to transfer concepts to market acceptable products. Scientists are often the concept providers, and the paint (or chemical) industry the product producers and market providers. Proof of concept is the bridge
11.2 A development model. All horizontal boxes represent concepts and the critical process when concepts must be transferred into products. Within that process, horizontal boxes must relate to the vertical boxes complying with regulatory and market demands in an interactive way. That process is best described as ‘proof of concept’ and it is critical in the development process. A hindrance may be different opinions in what is needed and what has to be proven. That may hamper, or in the worst case, put an end to the process.
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between the two. It should contain field efficacy and a basic set of toxicological testing, preferably at least degradation, bioaccumulation, cell toxicity and assurance that the suggested substance does not induce mutagenecity or bind to DNA-molecules. From my point of view, being a concept provider also includes assurances that the new substance may be suitable as a new antifoulant, and also have no mutagenic or bioaccumulative properties. Proof of concept is the most critical step associated with high risks in the innovation process and is not regularly discussed, and in some cases is referred to ‘not being my problem’. A functional communication triad is necessary to overcome problems associated with proofs of concept; to build collaboration linking concepts into products.
11.3.1 There is no single solution The biological complexity of the phenomenon we call biofouling is enormous. It is an ecological community with entities originating from all that we call life. Also, each organism has its own solution for how to find and stay firm on a surface, evolved during millions of years. In my view, it is impossible to invent new antifouling substances without restricting the problem, meaning that new molecules have to be part of a bigger solution. Any new substance will have an efficacy profile that differs from barnacles compared to algae (Konstantinou and Albanis, 2004). Therefore, it seems to be more logical and rational to use a combination of different substances to reduce the overall usage of marine biocides. By being more precise in mode of action, identifying key biological components in the biology of surface attachment, it is possible to reduce the amount of the biocide.
11.3.2 Rational development needs multidisciplinary action: the barnacle example As all organisms have their own solutions in adhering, we may also find several new ways to combat marine fouling and it is likely to be driven forward by increased knowledge in relevant sciences. There is a need to understand the general fouling biology, an increased knowledge in the area between traditional marine biology and surface chemistry. In our experience, based on cyprid larvae of the species Balanus improvisus, it is not difficult to demonstrate antifouling effects in laboratory of most substances in concentrations about 1 μM and above (Rittschof et al., 2003; Dobretsov and Qian, 2003; Dahlström et al., 2005). Without confirming a mode of action, this is not satisfactory from a scientific point of view. It points towards the fact that to understand attachment mechanisms, it is not enough to perform experiments with pharmacological molecules, without knowing if the biological targets exist in barnacles or other fouling organisms. It has
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to be combined with a number of different assays and experimental approaches (see Chapter 12: Dahms and Hellio).
11.4
Learning from the pharmaceutical industry
A focus within the pharmaceutical industry has always been to know the physiology as well as pathology within a chosen area. To be able to develop a pharmaceutical against high blood pressure, it is essential to identify hormones and neurotransmitters that contribute to blood pressure regulation and their specific receptors. Especially the receptors have proved to be fruitful targets in the development of small molecules that could alter the physiological output in a more favourable way. When a receptor, or ion channel or enzyme, has been identified as a key target for pharmaceutical treatment, it is then possible to screen for new substances in a systematic way. With molecular techniques, it is possible to isolate and develop biological assay with the target protein, a receptor of a specific hormone or neurotransmitter. With the isolated protein, expressed in a bioassay, the chemists will be able to create a vast number of derivate molecules which become a chemical library that can be tested against the target protein within an appropriate bioassay. In such a way, it is possible to create a high throughput system that combines knowledge and serendipity. The development that has made this possible is most of all knowledge in basic physiology and the ability to adopt modern technology within test systems that could become new bioassays. This methodology is also a possibility within antifouling research. An objection is that there are too many and too different types of organisms regarding marine biofouling so that it would be impossible to introduce such systematic research routes. However, the most basal physiological systems are similar between species. It is possible to identify targets and perform high throughput systematic research using general systems such as neurotransmitter receptors. Barnacles are no different from most other organisms. They do have neurotransmitters such as dopamine (Okano et al., 1996), serotonin (Yamamoto et al., 1996; Yamamoto et al., 1999), histamine (Callaway and Stuart, 1998), acetylcholine (Faimali et al., 2003) and probably also octopamine. Barnacle octopamine receptors have been identified and the receptor is known from other crustaceans. However, usually noradrenaline or adrenaline are not thought to act as neurotransmitters within invertebrates but are exclusive for vertebrate species (Roeder, 2005). There are some indications that noradrenaline may affect settling and metamorphosis (Yamamoto et al., 1996; Okano et al., 1996) in higher concentrations, further supported by antisettling effects seen with phentolamine (Yamamoto et al., 1998). However, phentolamine and yohimbine are also
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known as octopamine antagonists (Evans and Robb, 1993; Roeder, 2005; Maqueira and Chatwin, 2005) and noradrenaline binds as well towards dopamine receptors (Degen et al., 2000). Any results suggesting receptor specificity using the settling assay must be regarded with some care. The term specificity is a relative term based on mammalian pharmacology where the minutiae differences between receptor subtypes have been studied. Claiming specificity needs comparative studies between different receptor types. There are few such studies regarding invertebrate receptors and none within the area of antifouling. Instead, several adrenergic compounds bind to octopamine receptors (Evans and Robb, 1993; Howell and Evans, 1998) and dopamine receptor pharmacology within invertebrates is different compared to what is known from vertebrate, especially mammalian, studies. (Degen et al., 2000; Ödling et al., 2007; Zega et al., 2007). Any claim of specificity must be carefully evaluated on its own merits. What is lacking is knowledge regarding receptor biology and pharmacology, especially when looking at future rational development within the area. Only two receptors have been cloned (Isoai et al., 1996; Kawahara et al., 1997), both octopamine receptors (GPR18_BALAM (Q93127) and GPR9_BALAM (Q93126); www.expasy.org). A successful pharmacological approach must include detailed knowledge regarding receptor biology in terms of affinity, efficacy and specificity. This does not mean that we need to characterize every species. There are similarities between species even if science tends to emphasize the differences. As seen above, dopamine is a ubiquitous neurotransmitter and substances that have been developed for human diseases do affect barnacle cement secretion (Ödling et al., 2006). But to ensure that the right receptor protein is targeted, more biological assays need to be developed as well as molecular biology tools need to be applied in future developmental work. Regarding medetomidine as an example of a new antifoulant, the goal has been to find a reversible mechanism that does not necessary kill the cyprid larva, only prevent settling. We know that medetomidine binds to a receptor; the effect is reversible and specific antagonists have been identified (Dahlström et al., 2000; 2005). The mode of action is activating swimming movements that will short cut the surface behaviour (Hasselberg Frank, in manuscript). At the same time, the surface is nevertheless of great importance. Medetomidine is a surface active molecule (Dahlström et al., 2000; 2004). The receptor responsible for medetomidine activity within barnacles has now been cloned and been transferred into a yeast and been expressed within yeast cells (see Lind; 14th ICMCF conference, Kobe, Japan). This opens up the possibility to find and explore future substances that can be found within a chemical library that has the right biological and chemical properties that could either activate or block the receptor. In similar way,
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any identified receptor, for example a dopamine receptor, can be transferred over into an expression system and become the target in evaluating a chemical library designed for invertebrate dopaminergic receptors. The differences between pharmaceutical and antifouling development is the life cycle analysis of the biological active substance. To be included within a paint matrix or absorbed in the gastrointestinal system are two different means of distribution. A paint system may want a more lipophilic substance, whereas a more hydrophilic may be advantageous regarding a pharmaceutical. Degradability is a key issue within antifouling to avoid bioaccumulation, whereas a high degradation rate might not be favourable regarding pharmaceuticals, since it might mean a more complicated dosing regimen. If the biological target is identified and high throughput screening methodology implemented, it is possible to find substances that could meet biological efficacy criteria as well as having acceptable chemical properties. However, the ideal antifouling (or pharmaceutical substance) will never be found. There will always be compromises between the biological, chemical and environmental properties to decide between. Whether those decisions are right or wrong, that is the task for the regulatory system.
11.5
The importance of formulations
The paint industry has for centuries provided the market with different products and solutions. Most of the knowledge is empirical. Paint formulation is in many respects a difficult task, since small amounts of a new ingredient may totally change the physical and chemical properties. Rationally, it would be easier if incorporated biocides were a minor part of the formulation. At the same time, no one can foresee how mixtures may interact with each other and the formulation per se. The paint formulation is at the same time a storage and release system for the antifouling substance. As such, it plays a key role regarding efficacy and performance. In laboratory tests, medetomidine and a sister compound, clonidine are almost equally effective (Dahlström et al., 2000), but in field they differ totally. While medetomidine will stay completely effective for over two years, clonidine does not last for a single season on the Swedish west coast (unpublished data). This difference in efficacy is likely to be associated with surface behaviour of the molecules. Leakage of a new substance is one key issue when assessing environmental risks. Models like MAMPEC (free download from www.cepe.org) used to predict Predicted Environmental Concentration (PEC) is balancing between degradability, toxicity and leakage. A substance with low leakage rate is less sensitive to factors such as non-target toxicity or degradability. Leakage is also a parameter that can be refined by providing a control release system. A new tendency within paint formulation is to use
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microcapsule techniques also known from the pharmaceutical industry (Zhang et al., 2007).
11.6
Side effects and regulation
Dealing with such complex biological mechanisms, claiming efficacy and not expecting to find any side effect is a bit naïve. The question is more whether the side effects are acceptable and what the risks are with a specific substance. We may all conclude that every new antifouling substance will be hazardous, but the question we ought to answer is whether the risk is acceptable. In those terms, it is a great advantage if the mode of action is known. Is it a common biological mechanism that is a target and does it act through toxicity alone? Then the degradation data will become more critical, since general mechanisms are generally more hazardous. This is seen with substances such as Sea-Nine which has a high degree of toxicity but is quickly degradable and not bioaccumulative (Willingham and Jacobson, 1996; Jacobson and Willingham, 2000). The risks are therefore acceptable. Medetomidine is an example of the opposite way of dealing with side effects. It is specific and is fully reversible (Dahlström et al., 2000). Tests can be set up to predict foreseeable side effects from what is already known regarding its mode of action. The hazard is less, but still risks need to be quantified. Even though medetomidine is not hydrolyzed and degraded as quickly as Sea-Nine 211, it is not bioaccumulative (Hilvarsson, 2007) and its release rate is in ng/cm2/day (Dahlström, 2004; Borgert, 2004). Those features allow medetomidine to be used as an antifouling substance.
11.6.1 The regulatory process A way of handling risks from the society perspective is to have a regulatory procedure. Regarding Europe, this is now being transformed from the national level towards a European legislation. This is summarized in the Biocidal Product Directive (BPD) 98/8. For many European countries it changes the legislation regarding antifouling products. In most European countries notification of antifouling substances has been necessary but not a regulatory process. These issues are discussed in Chapter 10.
11.7
Marketing a new product
No development efforts are without a price, and antifouling development must in the end be economically sound. The product per se must be priced such that it may enter the market – but still not be underfinanced, since that will stop any further development. No one will ever say:
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We are prepared to take the costs for a new antifoulant.
In a broader perspective, this means that for a successful and a more dynamic market, we as scientists must learn more about issues beyond science if we are to be realistic in our hopes for our results and inventions. We must learn that to make a compound a success, it means more than efficacy in lab and field; it also means dealing with and judging the outcome from much more difficult aspects such as formulation, toxicological risks, as well as keeping on an eye on the market. In that process we need to interact with other experts, regulatory authorities as well as industrial competences. Once again, probably the most successful industry in terms of profit, the pharmaceutical sector, learned this lesson much earlier than the biocide sector. The sooner we create a more dynamic viewpoint, the more successfully will science come up with new possible and realistic alternatives that could interact with authorities and industry. As part of a three communication triad, industry and authorities need to have the ability and willingness to interact with new dynamic approaches and possibilities within science. When comparing the antifouling sciences with the pharmaceutical industry, one must also be aware of the differences between those industry sectors. Most of all, the pharmaceutical industry market is, in terms of money, much bigger than the antifouling paint market. It means that the driving forces and the risks that venture capitalists are willing to take are also higher. Therefore, pharmaceuticals are subsidized in many societies and therefore not comparable with the antifoulant market. It means that the innovation rate within the pharmaceutical industry can be faster due both to market size as well as society contribution measured as efforts and resources invested in science. Efforts are also driven by emotional factors since pharmaceuticals concern us as human beings. This is not the case regarding antifouling, although the increased awareness of the marine environment has been raised, also seen as new regulatory standpoints. Scientifically, the pharmaceutical and antifouling development processes are similar; find a substance, formulate and register. Although the overall biological complexity within the area of marine biofouling is overwhelming, especially knowing the lack of background information that exists in biomedicine and clinical sciences, as a matured industrial sector, pharmaceutical development serves as an example to reflect upon.
11.8
Summary
In many ways, antifouling research is still a very young scientific area, although efforts to protect marine surfaces have been an issue for mankind as long as the sea has been used for transportation. Our main and most
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used antifouling substance is still copper, used as an antifouling substance in paints since the 19th century. However, with the increased awareness of the marine environment and the new regulatory situation, this might change. There is an expectation that more substances will be banned and therefore create a demand for new ones with fewer risks, developmental work including academia, industry and society. Although, the trend can be the complete opposite due to the costs associated with the regulatory demands, causing new innovations to become economically impossible and therefore damage future opportunities for a better marine environment. In the long run, it might also be a dead end for market providers in that the alternatives will become fewer, reduced to few products with many producers. This is more of a political issue, but if we are serious in wanting new environmentally friendly products, we need all parts of the communication triad to share risks and development costs. In my veiw, new products are necessary to prove success for all parts involved, regardless of being concept or market providers.
11.9
References
Borgert, T. (2004). Investigation of interaction between medetomidine and alkyd using dynamic dialysis. Gothenburg: Chalmers University of Technology. Callaway, J. & Stuart, A. (1998). The distribution of histamine and serotonin in the barnacle’s nervous system. Microscopy Research and Techniques, 44 (2–3), 94–104. Dahlström, M. (2004). Pharmacological agents targeted against barnacles as lead molecules in new antifouling technologies. Gothenburg: University of Gothenburg. Dahlström, M., Jonsson, P. R., Lausmaa, J., Arnebrant, T., Sjögren, M., Holmberg, K. et al. (2004). Impact of Polymer Surface Affinity of Novel Antifouling Agents. Biotechnology and Bioengineering, 86 (1), 1–8. Dahlström, M., Lindgren, F., Berntsson, K., Sjögren, M., Mårtensson, L., Jonsson, P. et al. (2005). Evidence for different pharmacological targets for imidazoline compounds inhibiting settlement of the barnacle Balanus improvisus. Journal of Experimental Zoology, 303A (7), 551–562. Dahlström, M., Mårtensson, L., Jonsson, P., Arnebrant, T. & Elwing, H. (2000). Surface active adrenoceptor compounds prevent the settlement of cyprid larvae of Balanus improvisus. Biofouling, 16 (2–4), 191–203. Degen, J., Geweke, M. & Roeder, T. (2000). The pharmacology of a dopamine receptor in the locust nervous tissue. European Journal of Pharmacology, 396, 59–65. Dobretsov, S. & Qian, P.-Y. (2003). Pharmacological induction of larval settlement and metamorphosis in the blue mussle Mytilus edulis L. Biofouling, 19 (1), 57–63. Evans, P. & Robb, S. (1993). Octopamine receptor subtypes and their modes of action. Neurochemical Research, 18 (8), 869–874. Faimali, M., Falugi, C., Gallus, L., Piazza, V. & Tagliafierro, G. (2003). Involvement of acetyl choline in settlement of Balanus amphitrite. Biofouling, 19 (1), 213–220. Hilvarsson (2007). The antifoulant medetomidine. Sublethal effects and bioaccumulation in marine organisms PhD. thesis. University of Gothenburg, Dept of Marine Ecology.
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Howell, K. & Evans, P. (1998). The characterization of presynaptic octopamine receptors modulating octopamine release from an identified neurone in the locust. The Journal of Experimental Biology, 201, 2053–2060. Isoai, A., Kawahara, H., Okazaki, Y.-I. & Shizuri, Y. (1996). Molecular cloning of a new member of the putative G-protein-coupled receptor gene from barnacle Balanus amphitrite. Gene, 175 (1–2), 95–100. Jacobson, A. & Willingham, G. (2000). Sea-nine antifoulant: an environmentally acceptable alternative to organotin antifoulants. The Science of the Total Environment, 258, 103–110. Kawahara, H., Isoai, A. & Shizuri, Y. (1997). Molecular cloning of a putative serotonin receptor gene from barnacle, Balanus amphitrite. Gene, 184, 245–250. Konstantinou, I. & Albanis, T. (2004). Worldwide occurence and effects of antifouling paint booster biocides in the aquatic environment: a review. Environmental Inernational, 30, 235–248. Maqueira, B. & Chatwin, H. E. (2005). Identification and characterization of a novel family of Drosophila beta-adrenergic-like octopamine G-protein coupled receptors. Journal of Neurochemistry, 94, 547–560. Ödling, K., Albertsson, C., Russell, J. T. & Mårtensson, L. G. (2006). An in vivo study of exocytosis of cement proteins from barnacle Balanus improvisus (D.) cyprid larva. The Journal of Experimental Biology, 209, 956–964. Ödling, K. (2007). The cement gland of the Balanus improvisus cyprid larva – with special emphasis on secretion and regulations. Licentiate thesis, University of Gothenburg, Dept of Zoology. Okano, K., Shimizu, K., Satuito, C. & Fusetani, N. (1996). Visualization of cement exocytosis in the cypris cement gland of the barnacle Megabalanus rosa. Journal of Experimental Biology, 199 (10), 2131–2137. Rittschof, D., Lai, C.-H., Kok, L. M. & Teo, S. L.-M. (2003). Pharmaceuticals as antifoulants: Concept and principles. Biofouling, 19 (1), 207–212. Roeder, T. (2005). Tyramine and octopamine: Ruling behaviour and metabolism. Annual Review of Entomology, 50, 447–477. Willingham, G. & Jacobson, A. (1996). Designing an environmentally safe marine antifoulant. ACS symposium series, 640, 224–233. Yamamoto, H., Satuito, C., Yamazaki, M., Natoyama, K., Tachibana, A. & Fusetani, N. (1998). Neurotransmitter blockers as antifoulants against planktonic larvae of the barnacle Balanus amphitrite and the mussel Mytilus galloprovincialis. Biofouling, 13 (1), 69–82. Yamamoto, H., Shimizu, K. & Tachibana, A. F. (1999). Roles of dopamine and serotonin in larval attachement of the barnacle, Balanus amphitrite. Journal of Experimental Zoology, 284, 746–758. Yamamoto, H., Tachibana, A., Kawaii, S., Matsumura, K. & Fusetani, N. (1996). Serotonin involvement in larval settlement of the barnacle, Balanus amphtrite. The Journal of Experimental Zoology, 275, 339–345. Zega, G., Pennati, R., Dahlström, M., Berntsson, K., Sotgia, C. & De Bernardi, F. (2007). Settlement of the barnacle Balanus improvisus: The roles of dopamine and serotonin. Italian Journal of Zoology, 74 (4), 351–361. Zhang, M., Cabane, E. & Claverie, J. (2007). Transparent antifouling coatings via nanoencapsulation of a biocide. Journal of Applied Polymer Science, 105, 3824–3833.