The adhesion of the barnacle Elminius modestus (Darwin) to fouling-release coatings. Rebecca Martin Doctor of Philosophy in Marine Science School of Marine Science and Technology Newcastle University August 2017 Abstract The main aim of this thesis was to investigate the potential of Elminius modestus (= Austrominius modestus) for evaluating the performance of fouling-release (FR) coatings. A secondary aim was to explore how the membranous-basis of this species influences the fracture mechanics and release from FR coatings in comparison to Balanus amphitrite (= Amphibalanus amphitrite), a barnacle with a calcareous-basis and widely adopted as a model for antifouling and FR studies. The critical removal stress (CRS) − the force required to remove fouling organisms, normalised by contact area − is a standard measure to evaluate FR coatings using either barnacles with calcareous-bases or metal studs (‘pseudobarnacles’). Testing FR coatings against a diverse range of fouling organisms is necessary to evaluate the global effectiveness of a coating. The percentage settlement of cyprids, growth rate, and CRS of laboratory- cultured barnacles were evaluated on polydimethylsiloxane (PDMS) standard coatings (Silastic T-2 and Sylgard 184). The percentage settlement on the PDMS coatings between the two species did not significantly differ, however, there were differences in the growth rate and CRS. When grown on Silastic T-2 and Sylgard 184 and fed Tetraselmis suecica algae, E. modestus grew at a faster rate than that of B. amphitrite. There was also a significant coating effect on the growth of E. modestus with barnacles on Sylgard 184 growing to larger size than those grown on Silastic T-2. The CRS of E. modestus was less than that for B. amphitrite but only for the coating Sylgard 184. Using high-speed photography, the separation processes of E. modestus and B amphitrite, from Silastic T-2 and Sylgard 184 coatings was observed. Four distinct separation patterns were characterised; lift, peel, adjacent peel and twist. These were based on the location of the initial separation and direction of propagating instabilities in respect to the direction of detachment force. The observed differences in the separation patterns between species may have more to do with the variations in shape and structure of the barnacle shell than to the type of basis. However, the flexibility of the membranous-basis of E. modestus was important for the propagation of the fracture as it hindered the formation of fingering instabilities as they progressed through the adhesive interface. i The bulk properties of five polysiloxanes and three fluoropolymers were modified by changing the polymer chain length and cross-linker density, which provided coatings with a modulus ranging from 0.31 to 19.73 MPa. These were used to investigate whether laboratory assays were a good predictor of a coatings performance in the field, in terms of settlement/recruitment and CRS. Two field populations (Fairlie Quay and Burnham-on-Crouch) over two years (2010 and 2011) were compared to a laboratory culture of E. modestus barnacles. There were similarities between the laboratory settlement/field recruitment and CRS of E. modestus from the two field populations and the laboratory culture across the eight coatings. This made it possible to discriminate between the coatings. Although, the CRS measurements did significantly differ between locations and years, where the general pattern from highest to lowest in terms of CRS between the locations was Fairlie Quay > laboratory > Burnham-on-Crouch. These eight coatings were also used to investigate the degree in which the elastic modulus of a coating can influence the CRS of E. modestus, compared to the CRS of B. amphitrite. The regression analysis confirmed that as the modulus increases the CRS for both species increases. There were marked differences in the removal of barnacles from the high modulus fluoropolymers. B. amphitrite, unlike E. modestus, failed to detach and left the basis on the coating’s surface. As E. modestus can differentiate between the coatings in terms of FR efficacy and was amenable to laboratory culture with a comparable growth rate to B. amphitrite, this species is recommended as an additional model for FR studies. ii Acknowledgements I wish to express my appreciation and gratitude for everyone who has helped throughout my PhD years. Firstly I would like thank my primary supervisor Prof. Tony Clare. Without your support and help I wouldn’t have had the opportunity to embark on this PhD. But also for your excitement, guidance and tolerance throughout the years. I would next like to thank my second supervisor Dr. Gary Caldwell. I truly appreciate all your dedication and your encouragement over my project especially with my written work. There are also my Industrial supervisors Jennifer Longyear (June 2010 – January 2013) and Gabrielle Prendergast (January 2009- June 2010). Thank- you both for your enthusiasm and support. I would like to gratefully acknowledge International Paint Ltd for supporting this studentship. There are many within International Paint who have contributed their time and expertise, helping me to produce the coatings, characterise the coatings, helping me to understand the chemistry of the coatings and with the field-work at Burnham-on-Crouch. In particular I would like to thank David Williams, David Stark, Cait Davies, Kevin Reynolds, Graeme Lyall, Adam Bell, Lyndsey Tyson, Trevor Wills and Simon Kelly. There are also the kind people at Fairlie Quay to thank, who were more than happy to allow me to tie my panels to their pier for three years. My greatest appreciation to Sheelagh Conlan and Nick Aldred for their expert knowledge on culturing barnacles and techniques on settlement and adhesion testing. Thank you to the technicians David Whitaker, Ali Trowsdale, John Rand in the Ridley Building and John Knowles in the Dove Marine Laboratory. For all their help, including but not limited to aquarium and laboratory maintenance, and help in producing algae and setting-up equipment. I would also like to say a big, BIG thanks to all my fellow PhD students Helen G, Supanut, Alessio, Susan, Hilary, Sofia, Thea for the “insightful” debates over much needed tea and biscuits. Last but by no means least, thank you to my husband Ross and my mother Janet. You have both made my PhD years possible by being a constant comfort and lovingly supportive. And a special thanks to my mother, for all your help proof reading and correcting my terrible grammar. But also for knowing the need of any student for chocolate, tea (and wine) and more biscuits. iii iv Contents Page number Abstract i Acknowledgements iii Contents v List of Figures x List of Tables xviii Abbreviations xxiii Chapter 1. Development of a Test Species for Fouling-Release 1 Research: An Introduction. 1.1. Introduction 1 1.2. Biofouling 2 1.3. Elminius modestus: An introduction 6 1.4. Antifouling 12 1.5. Tributyl tin (TBT) 14 1.6. Alternative antifouling paints 15 1.6.1. Novel alternatives 16 1.6.1.1. Micro-topography 16 1.6.1.2. Natural products 17 1.6.1.3. Enzymes 17 1.7. Fouling-release coatings 18 1.7.1. Surface energy 19 1.7.2. Elastic modulus and thickness 21 1.7.3. Chemical composition of fouling-release coatings 24 1.8. Recent developments in fouling-release research 25 1.9. Methods for assessing fouling-release coatings 27 1.9.1. Pseudobarnacles 27 1.9.2. Choice of marine organisms 28 1.9.3. Barnacles 33 1.9.3.1. Laboratory culture of barnacles 35 1.9.3.2. Field immersion trials 36 1.10. Critical removal stress of barnacles 38 1.10.1. Critical removal stress of adult barnacles 38 1.10.2. Removal stress of cyprids 39 1.11. Research Gap 39 1.11.1. Elminius modestus: As a test species 41 1.12. Thesis objectives 41 v Chapter 2: An Assessment of Elminius modestus (Darwin) - 43 a Barnacle with a Membranous-Basis - as a Model Species for Evaluating Fouling-Release Coatings. 2.1. Abstract 43 2.2. Introduction 44 2.3. Materials and methods 45 2.3.1. Coating preparation 45 2.3.2. Maintenance of adult barnacles 46 2.3.3. Larval culture 47 2.3.3.1. Elminius modestus 47 2.3.3.2. Balanus amphitrite 48 2.3.4. Influence of the culture medium on the settlement of 48 Elminius modestus 2.3.5. Settlement assays 49 2.3.5.1. 24-well plate assays 49 2.3.5.2. Settlement on coated surfaces 49 2.3.6. Growth measurements 50 2.3.7. Critical removal stress measurements 51 2.3.7.1. Influence of size of Elminius modestus on 51 the critical removal stress 2.3.7.2. The critical removal stress of Elminius 52 modestus and Balanus amphitrite 2.3.8. Statistical analysis 52 2.3.8.1. Laboratory settlement assays 52 2.3.8.2. Growth 52 2.3.8.3. Critical removal stress 53 2.4. Results 53 2.4.1. Influence of the culture medium on the settlement of 53 Elminius modestus 2.4.2. Settlement of Elminius modestus and Balanus 55 amphitrite 2.4.3. Growth 58 2.4.4. Critical removal stress measurements 62 2.4.4.1. Influence of size of Elminius modestus on 62 the critical removal stress 2.4.4.2. A comparison of critical removal stress of 64 Elminius modestus and Balanus amphitrite 2.5. Discussion 65 2.5.1. Influence of the culture medium on the settlement of 66 Elminius modestus 2.5.2. Settlement of Elminius modestus and Balanus 68 amphitrite 2.5.3. Growth 69 2.5.4. Critical removal stress 71 2.6. Conclusion 73 vi Chapter 3. High-Speed Video Analysis of the Detachment of 75 Barnacles with Membranous and Calcareous Bases.