Biofouling The Journal of Bioadhesion and Biofilm Research ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/gbif20 Using encrusting bryozoan adhesion to evaluate the efficacy of fouling-release marine coatings G. T. Waltz , K. Z. Hunsucker , G. Swain & D. E. Wendt To cite this article: G. T. Waltz , K. Z. Hunsucker , G. Swain & D. E. Wendt (2020): Using encrusting bryozoan adhesion to evaluate the efficacy of fouling-release marine coatings, Biofouling, DOI: 10.1080/08927014.2020.1857742 To link to this article: https://doi.org/10.1080/08927014.2020.1857742 Published online: 20 Dec 2020. Submit your article to this journal Article views: 10 View related articles View Crossmark data Full Terms & Conditions of access and use can be found at https://www.tandfonline.com/action/journalInformation?journalCode=gbif20 BIOFOULING https://doi.org/10.1080/08927014.2020.1857742 Using encrusting bryozoan adhesion to evaluate the efficacy of foulingrelease marine coatings G. T. Waltza, K. Z. Hunsuckerb , G. Swainb and D. E. Wendta a Center for Coastal Marine Sciences, Cal Poly, San Luis Obispo, USA; bCenter for Corrosion and Biofouling Control, Florida Institute of Technology, Melbourne, USA ABSTRACT ARTICLE HISTORY Biofouling communities are spatiotemporally diverse, underscoring the need to assess foulingrelease (FR) coating performance against common biofouling taxa at multiple field sites. Adhesion strength assessments of FR coatings incorporate few taxa into standardized protocols. This study tested the feasibility of incorporating existing ASTM barnacle protocols on tubeworms and encrusting bryozoans (EB). Additionally, trends in adhesion strength among these taxa were compared at two field sites. EB adhesion at both field sites showed consistent results and adhesion strength followed the same trend: tubeworms > barnacles >EB. Testing EB adhesion was feasible and enhanced assessments of FR coatings by increasing the diversity of assessed taxa. Received 9 June 2020 Accepted 23 November 2020 Introduction Biofouling is the accumulation of organisms on human-made (e.g. ships’ hulls, pier pilings) and natural surfaces in the ocean. Biofouling can negatively impact many anthropogenic activities and includes both economic as well as ecological impacts (Lewis 1998 and Chambers et al. 2006). For example, biofouling can cause a reduction in ship fuel efficiency due to increased drag through the water (Redfield and Hutchins 1952; Schultz 2007), reduce water flow through plumbing (Redfield and Hutchins 1952), physically damage structures immersed in the ocean (Redfield and Hutchins 1952), and function as a mechanism for the spread of invasive species (e.g. D€ urr and Thomason 2009). Predicting and managing biofouling can be difficult due to the spatial and temporal variability of these communities (e.g. Sutherland and Karlson 1977; Holm et al. 2000; Swain 2017). Ships traveling globally may be exposed to varied fouling communities from tropical and polar seas on a relatively short timescale. Also, ships within a given ecoregion may experience seasonal fluctuations in community composition. The aforementioned variability inherent to biofouling communities will likely be further impacted by changes in the global climate and oceanographic conditions. Community level changes to marine ecosystems were recorded during recent large-scale ocean perturbations (e.g. Cavole CONTACT Grant Waltz gwaltz@calpoly.edu ß 2020 Informa UK Limited, trading as Taylor & Francis Group KEYWORDS Adhesion strength; biofouling; bryozoans; fouling-release coatings et al. 2016; Dobretsov et al. 2019). Of note, shifts in biofouling communities were recorded from the recent marine heat wave in 2014 and the 2015 2016 El Ni~ no along the west coast of North America (Wendt and Waltz, unpublished data). Some of the frequently encountered taxa in biofouling communities across the globe includes encrusting bryozoans, barnacles, and tubeworms. They have been shown to be abundant in fouling communities in both the Pacific and Atlantic and their persistence as fouling organisms can lead to broad economic and ecological impacts (Swain et al. 2000; Wood et al. 2000; Hearin et al. 2015; Bastida-Zavala et al. 2017). Given the economic and ecological impacts from biofouling of human-made structures and the diversity of species in fouling communities across the globe, a major effort has been made to produce nontoxic foul-release (FR) coatings, effective against a broad suite of fouling organisms (Swain 1999). Static immersion sites are used to subject coatings to real world conditions and assist in the down-select process of new coatings through a suite of field testing procedures (Swain and Schultz 1996). By exposing the same coatings to a variety of oceanic conditions and biofouling communities at multiple test sites, it is possible to obtain a more complete assessment of coating performance than if a single test site were utilized (e.g. Swain et al. 2000; Holm et al. 2006). 2 G. T. WALTZ ET AL. Due to differences in coating formulations and adhesion strategies in fouling organisms, performance of coatings is not spatially consistent and varies by biofouling taxonomic group (Holm et al. 2000, Zargiel et al. 2011). Stein et al. (2003) showed there was an interaction between coating type and fouling species; coatings that worked well for one species did not necessarily work as well for another species. Thus, there is also a need to assess coating performance against multiple species during the coating development and testing process. The commonly tested group of species should include those that are ubiquitous across test sites. Testing novel coatings at spatially discrete test sites provides coating developers with more robust information about the expected coating performance, since the coatings are exposed to a greater variety of fouling taxa and the subsequent oceanic conditions that affect these taxa. Fouling taxa are broadly divided into two categories: hard-fouling and soft-fouling (Callow and Callow 2002). Hard-fouling taxa are characterized by rigid external structures (e.g. barnacles, calcareous tubeworms, encrusting bryozoans) while soft-fouling taxa lack this rigid structure (e.g. biofilms, algae, tunicates, anemones). Standardized methods have been developed to test the effectiveness of FR coatings against both categories of fouling taxa (recently reviewed by Wendt 2017). Swain et al. (1992) developed methods to assess adhesion strength, in shear, of barnacles (ASTM D5618 ). The adhesion strength or critical removal stress (CRS) is a metric for quantifying how strongly sessile hard-fouling organisms are attached to the substratum and has been used to grade the performance of developmental FR coatings (Wendt et al. 2006). Despite the diversity of organisms comprising the hard-fouling category, only barnacles have been included in the ASTM protocol, which assesses the adhesion strength on FR coatings. While barnacles are a globally diverse and abundant taxa, there are few studies assessing the adhesion strength of barnacles relative to other globally abundant and diverse hard fouling taxa (Wood et al. 2000; Kavanagh et al. 2001). While barnacles can provide valuable data they are often not the dominant hard-fouling organism present at static immersion sites, and are often outcompeted by colonial organisms. For example, encrusting bryozoans (EB) compose > 40% of the fouling community at the static immersion test site in California during certain times of the year (Wendt, unpblished data) and was shown to be one of the major fouling groups in both Hawaii (Wood et al. 2000) and Florida (Hearin et al. 2016). In an effort to broaden the hard-fouling taxa that can be used for screening coatings at static field sites, the efficacy of using EB as a metric for FR properties was tested in comparison with the barnacle species which are currently employed (ASTM D5618-20 2020) and calcareous tubeworms which have previously been reported in the literature (Kavanagh et al. 2001). Specifically, two questions were addressed in this study (1) Could valid data be acquired for FR coatings by testing the adhesion strength of EB using the ASTM standard, and (2) how did the adhesion results from the broad taxonomic categories ‘calcareous tubeworms’, ‘encrusting bryozoans’, and ‘barnacles’ compare from two spatially removed field test sites? Material and methods Test sites The adhesion strength of the fouling organisms mentioned above was assessed at two static immersion test sites, one in California and the other in Florida. The California Polytechnic State University, San Luis Obsipo (Cal Poly, USA) static immersion test site in Morro Bay, California, is located approximately midway between Los Angeles and San Francisco. The static immersion test site is situated in a tidally influenced estuary and has a biofouling community dominated by barnacles, EB, colonial tunicates, and hydroids (Needles and Wendt 2013). This field site is in a temperate marine environment under the influence of periods of upwelling. Water temperature fluc tuates between 10 16 C, with salinity ranging from 13 35 ppt. The Florida Institute of Technology (FIT, USA) static immersion test site is located in Port Canaveral, along the central east coast of Florida. The port was created in 1953 and is a hub for cruise and cargo ships, US Navy, Coastguard, fishing vessels and recreational boats. It is an area of high fouling activity. The biofouling community is dominated by tubeworms, barnacles, encrusting bryozoans, and colonial tunicates. This is a subtropical environment and the   water temperature fluctuates between 20 C and 32 C, with an average salinity of 35 ± 1.2 ppt (Hunsucker et al. 2019). Marine coatings Formulations of commercially available and US Navy approved silicone based FR coatings were applied to metal or PVC panels (101 mm  203 mm) and immersed in the ocean 0.5 1.0 m below the water BIOFOULING surface. Coatings were manufactured by International Paint (Akzonobel, UK) and are known as the Intersleek series of FR paints. All coatings were applied according to the manufacturer’s specifications, with a top coat of 200 mm in thickness. Cal Poly tested adhesion strength on panels coated with the Intersleek 757 (silicone elastomer FR coatings) and Intersleek 970 (fluoropolymer FR coatings). Due to coating availability and differences in settlement, barnacle and EB adhesion strength were assessed relative to each other on Interlseek 757, with tubeworm and EB adhesion assessment on Intersleek 970. FIT tested adhesion on panels coated with the Intersleek 970 for all three taxa. Adhesion test taxa Barnacle Barnacles are crustaceans with a sessile, filter feeding adult stage. Barnacles from the suborder Balanomorpha have a hard calcareous exterior shell system made of plates and a hard calcium carbonate basal plate (Carlton 2007). The basal plate adhesive is proteinaceous. The basal plate is calcified in all species within Balanomorpha (Carlton 2007). Planktonic larval barnacles undergo six molts and then metamorphose to the terminal stage, the cypris larva. The cyprid is non-feeding and undergoes settlement, attachment to a suitable substratum, and then metamorphosis to a sessile juvenile barnacle. There have been extensive studies into the mechanism of the adult adhesive process as well as the physical composition of the adult barnacle adhesive (e.g. Burden et al. 2012; Kamino 2013; Daugherty 2016). In general, liquid proteinaceous adhesives are secreted, most likely in more than one step, by the barnacle which then solidifies onto the substratum (Burden et al. 2012). Three species of barnacles were tested during this study. At Cal Poly Balanus crenatus were assessed and at FIT Amphibalanus amphitrite and A. eberneus were assessed. EB colonies Adult encrusting bryozoans (EB) are marine, sessile, colonial, filter feeding invertebrates. Adult bryozoans generally brood embryos until they are ready for release into the plankton though there are some species that release gametes directly into the water column. Larval bryozoans can be either feeding or nonfeeding, depending on the species and can swim through the water using cilia (Brusca et al. 2016). When preparing to settle, larval bryozoans use a 3 sensory structure (vibratile plume) to locate a suitable substratum for settlement. Once settled they undergo metamorphosis into an ancestrula (Woollacott and Zimmer 1977; Price et al. 2017). The mechanisms of adhesion to the substratum for adult encrusting bryozoan colonies have not been well studied in many species. The limited previous work on similar species showed that EB colonies attach to the substratum with a protein/carbohydrate compound (Loeb and Walker 1977; Soule and Soule 1977). Several species of EB were tested during this study. At Cal Poly these were Celloporaria brunnea, Cryptosula pallisiana, and Membranipora sp. At FIT one species was present, Watersipora cf. subvoidea. Tubeworms Adult tubeworms from the family Serpulidae are sessile, filter feeding annelid worms that secrete a calcium carbonate tube that is adhered to the substratum (Brusca et al. 2016). Larval tubeworms use bacterial biofilm related cues to initiate settlement and begin the process to form the calcareous external tube (e.g. Nedved and Hadfield 2008).The adhesive material for serpulid worms has been examined in some detail (Tanur et al. 2010). The adhesive tube layer incorporates a similar aragonite and magnesium calcium crystal structure as the rest of the tube but also incorporates an organic sheet and fiber matrix (Tanur et al. 2010). Tubeworms tested at both sites were from the genus Hydroides but were different species. At Cal Poly, H. gracilis was assessed and at FIT, H. elegans was assessed Adhesion testing Panels were immersed at the respective field sites. They were checked and ‘gardened’ regularly for individual barnacles, tubeworms and, EB colonies. ‘Gardening’ was a process where organisms for adhesion testing were isolated. This was accomplished by clearing the coating around the selected organism, removing other fouling organisms not needed for testing (e.g. colonial tunicates and hydroids) and allowing the unimpeded growth of the adhesion organisms (e.g. barnacles). Individual barnacle, tubeworm, and EB colony CRS was determined following the methods of Swain et al. 1992 which were turned into a standard method (ASTM D5618). All removal force measurements were made using a handheld Imada ZTS 11 force gauge at Cal Poly and a Shimpo digital handheld force gauge at FIT. The removal process varied slightly by taxonomic group and field site 4 G. T. WALTZ ET AL. due to variations in growth morphology. See below for more detail. After removal each organism was placed into a compartmentalized container for transport to the laboratory where they could be measured, counted, and properly identified. The number of taxa analyzed during this study was dependent on the location and the coating. Panels remained in the water for over a year in order to obtain a proper sample size (n > 10). For most of the organisms tested, this time frame allowed for adequate growth and testing and an ‘n’ much higher than ten. Barnacles Individual barnacles were manually removed from coatings when the basal plate was between 5 mm 10 mm in diameter, as measured with a pair of dial calipers. Barnacles were pushed at the lowest possible point at the basal plate, placing the hand-held force gauge parallel to the coating surface. After removal, the amount of basal plate remaining (BPR) was visually estimated as the percentage total of the initial area of the basal plate. Individuals with > 10% BPR were excluded from the CRS analyses as per ASTM D5618. The area of the attachment surface (basal plate) was calculated from the diameter of the attachment surface measured using dial calipers in the field (Cal Poly) or through the Image J photo analysis software in the lab (FIT) (Schneider et al. 2012). CRS was determined by dividing the force at which the barnacle was removed from the coating surface by the area of the basal plate. EB colonies EB colonies were manually removed from the panel when they were between 5 mm 20 mm in diameter and measured, using the same technique as was utilized for barnacles. At the FIT test site, EB often grew to completely cover or encrust the test surface. When this occurred, the EB would be manually reduced in size using a blunt object (such as the head of a roofing nail) until a section remained that was 26.9 ± 5.5 mm in length. It was found that the thicker colonies were easier to isolate and manually remove with a force gauge. At times, calcareous tubeworms (Hydroides spp.) were found to grow on top of the EB colonies but the tubeworm had no contact with the actual FR coating. In this case, the tubeworm was used to assist in the push-off of the EB, such that the tubeworm was pushed which resulted in the tubeworm/EB colony releasing together. These tubeworm/ EB isolations were compared with EB isolations, and there was no significant difference in adhesion strength (p < 0.05), and thus both types could be used for assessing the adhesion on a FR coating. At Cal Poly, the EB isolation and pushing process varied slightly due to differences in colony growth morphology. EB were gardened until reaching a diameter of 5 mm 20 mm and were then pushed directly from the coating surface using a handheld force gauge. After removal, the amount of colony area remaining (CAR) was visually estimated as the percentage total of the initial area of the colony attachment surface. Individuals with >10% CAR were excluded from CRS analyses as per ASTM D5618. The area of the attachment surface of the entire colony was calculated from the diameter of the attachment surface measured using dial calipers in the field (Cal Poly) or through the Image J photo analysis software in the lab (FIT). CRS was determined by dividing the force at which the EB colony was removed from the coating surface by the area of the colony attachment surface. Tubeworms Individual tubeworms were manually removed from the panel when the calcareous tube was wider than 1.5 mm. Tubeworms were additionally trimmed such that the pushed tube was a straight line with a consistent width along the entire length of the tube. A framing nail or straight blade screw driver was used to trim narrow or curved tube sections before pushing was initiated. Tubeworms were pushed parallel to the long edge of the calcareous tube (e.g. into the opening of the tube) as opposed to perpendicular to the long edge of the tube. After removal, the amount of tube attachment surface remaining (TASR) was visually estimated as the percentage total of the initial area of the tube attachment surface area. Individuals with > 10% TASR were excluded from CRS analyses as per ASTM D5618. The area of the tube attachment surface was calculated by measuring the length and width of the pushed tube section in the field using dial calipers (Cal Poly) or with Image J (FIT). CRS was determined by dividing the force at which the tubeworm was removed from the coating surface by the area of the attachment surface. Statistical analysis CRS among tubeworms, barnacles, and encrusting bryozoans was analyzed for comparison with each other at each site using a one-way ANOVA in JMP Pro 14.3 (SAS, Cary, NC, USA). The alpha level was set at  0.05. Where significant differences were detected, post hoc analyses were conducted using a Tukey HSD test. Data were natural log transformed BIOFOULING 5 Table 1. ANOVA adhesion strength results from Cal Poly. Source DF Sum of squares Mean square F Ratio Prob > F Model Error C. Total 3 73 76 4.34092 17.25756 21.59848 1.44697 0.23640 6.12070 0.0009 The adhesion strength among EB (Celloporaria brunnea, Cryptosula pallisiana, and Membranipora sp.) and a barnacle (Balanus crenatus) on Intersleek 757. a was set at  0.05. There was a statistically significant difference in adhesion strength among the four species. Figure 1. Mean CRS and sample size for EB (Celloporaria brunnea, Cryptosula pallisiana, and Membranipora sp.) and barnacles (Balanus crenatus) tested at Cal Poly on Intersleek 757. Groups that share a letter do not have statistically significant differences in CRS. There was a statistically significant difference in CRS between the EB species C. brunnea and the EB species Membranipora sp. and the barnacle species B. crenatus. Error bars represent one standard error of the mean. to meet the normality and equality of variance assumptions of ANOVA. Results Adhesion on encrusting bryozoan colonies During the course of the experiment, several EB species were observed to settle on the FR coatings and there was variation in the species that comprised the fouling communities at both test sites. One species was present at FIT (Watersipora cf. subvoidea). Cal Poly tested three EB species (Celloporaria brunnea, Cryptosula pallisiana, and Membranipora sp.). These species varied in both morphology and overall coverage on the FR coatings. At Cal Poly, the EB species and subsequent total colony size were typically smaller, when pushed from the panel, than those reported at FIT because of the speed that the colonies grew and the colony morphology at the time they were pushed. At Cal Poly, the colonies could reach an appropriate diameter for testing in two to four weeks whereas at FIT EB colonies could cover the panel in about four weeks. C. brunnea grew to be 100 mm in diameter, was brown in coloration, relatively thick (up to 2 mm), formed irregular shaped colonies that were rough in texture, and would grow into complex three dimensional structures protruding up to 5 mm from the panel surface. C pallisiana colonies were bright orange, covering on average a 60 100 mm area, thinner than C. brunena (1 mm), were generally circular, grew low onto the panel surface (1 mm), and rarely formed threedimensional colonies. Membranipora sp. produced thin, flexible colonies that were a tan color, could cover > 100 mm of the panel, did not form threedimensional colonies, and grew low onto the panel surface (1 mm). Adhesion varied among the three EB species. On the FR coating IS757, C. brunnea had a non-transformed CRS of 0.046 ± 0.005 MPa, C. pallisiana had a non-transformed CRS of 0.076 ± 0.012 MPa, and Membranipora sp. a nontransformed CRS of 0.083 ± 0.007 MPa (Figure 1). There was a significant difference in the adhesion between the Membranipora sp. and C. brunnea (Table 1). However, the same two species also recruited to the IS900 coating, but with an insignificant variation in adhesion (Figure 2). Membranipora sp. had a nontransformed CRS of 0.078 ± 0.005 MPa and C. brunnea had a non-transformed CRS of 0.085 ± 0.011 MPa. At FIT, W. cf. subvoidea was a dominant member of the fouling community. It is a calcified encrusting bryozoan and living specimens are bright orange-red. W. cf. subvoidea could grow to completely encrust the whole (101 mm x 203 mm) panel. When this occurred, care had to be taken to isolate small sections for adhesion. The thickness of the W. subvoidea could also vary and reach up to about 3.2 mm. It was found that a minimum thickness of about 0.53 mm was best for isolating and overall adhesion testing. Over the course of testing, the non-transformed CRS of W. cf. subvoidea was 0.030 ± 0.010 MPa on the IS900 FR coating (Figure 3). Adhesion among hard fouling species In addition to the EB species discussed above, both barnacles and calcareous tubeworms were important members of the fouling community during this study. Barnacle species present were Balanus crenatus (Cal Poly) and Amphibalanus amphitrite and A. eberneus (FIT). The calcareous tubeworms were from the genus Hydroides, but varied in species based on location. H. 6 G. T. WALTZ ET AL. Table 2. ANOVA adhesion strength results from Cal Poly. Source DF Sum of squares Mean square F Ratio Prob > F Model Error C. Total 2 90 92 7.10100 30.09477 37.20472 3.5550 0.33439 10.63130 <0.0001 The adhesion strength among EB (Membranipora sp. and Celloporaria brunnea) and tubeworms (Hydroides gracilis) on Intersleek 970. a was set at  0.05. There was a statistically significant difference in adhesion strength among the three species. Figure 2. Mean CRS and sample size for EB (Membranipora sp. and Celloporaria brunnea) and tubeworms (Hydroides gracilis) tested at Cal Poly on Intersleek 970. Groups that share a letter do not have statistically significant differences in CRS. There was a statistically significant difference in CRS between both EB species and the tubeworm species. Error bars represent one standard error of the mean. Figure 3. Mean CRS and sample size for EB (Watersipora subvoidea), barnacles (A. amphritrite/eberneus) and tubeworms (Hydroides sp.) tested at FIT on Intersleek 970. Groups that share a letter do not have statistically significant differences in CRS. There were statistically significant differences in CRS among all three taxa. Error bars represent one standard error of the mean. gracilis was present at Cal Poly, with H. elegans dominating at FIT. At Cal Poly, differences in the adhesion strength among organisms were observed on both the IS757 and IS900 coatings. There was a statistically significant difference in CRS between barnacles and the EB species tested on IS757 (Figure 1, Table 1). Specifically, C. brunnea (non-transformed 0.046 ± 0.005 MPa) was significantly easier to remove than the barnacle B. crenatus (non-transformed 0.101 ± 0.008 MPa). However, the other two EB species, Membranipora sp. and Cryptosula pallasiana, did not show a statistically significant different CRS than B. crenatus. There was a general trend of decreasing CRS in the three EB species tested at Cal Poly, relative to B. crenatus (Figure 1). On IS900, tubeworms (Hydroides gracilis) and two of the EB species were present (Figure 2). H. gracilis (non-transformed 0.149 ± 0.013 MPa) had a statistically significantly higher CRS than Membranipora sp. (non-transformed 0.078 ± 0.005 MPa) and Celleporaria brunnea (nontransformed 0.085 ± 0.011 MPa) (Figure 2 and Table 2). Membranipora sp. and Celloporaria brunnea did not have a statistically significantly different CRS from each other (Table 2). In general, at Cal Poly there appeared to be a trend with regard to fouling organism adhesion: tubeworms had the highest CRS, then barnacles, and then EB. At FIT, all three hard fouling taxa had a statistically significantly different CRS on the IS900 FR coating (Figure 3 and Table 3). As with Cal Poly, there was a decreasing trend in the adhesion strength of taxa where tubeworms had the highest CRS, then barnacles, and EB had the lowest CRS. CRS for these taxa was as follows: Hydroides sp. (non-transformed 0.190 ± 0.090 MPa), A. amphitrite/eberneus (non-transformed 0.080 ± 0.040 MPa), and Watersipora subvoidea (non-transformed 0.030 ± 0.010 MPa, Table 3 and Figure 3). Discussion EB colonies were successfully incorporated into the existing ASTM standard for assessing the FR properties of marine coatings (ASTM D5618). The data collected on the EB colonies were comparable at both field sites with respect to the variability in the adhesion measurements and the feasibility of conducting the adhesion testing. The size and morphology of the EB colonies varied based on the species, but all were able to be isolated and analyzed for CRS data. In BIOFOULING Table 3. ANOVA adhesion strength results from FIT. Source DF Sum of squares Mean square F Ratio Prob > F Model Error C. Total 2 218 220 67.86770 64.34877 132.21647 33.93380 0.29520 114.96070 <0.0001 The adhesion strength among EB (Watersipora subvoidea), a barnacle (A. amphritrite/eberneus) and tubeworms (Hydroides sp.) on Intersleek 970. a was set at  0.05. There was a statistically significant difference in adhesion strength among all three groups. general, the EB adhesion ranged from 0.020 to 0.083 MPa. Adult EB formed sheet like growths composed of calcium carbonate that could grow vertically from the substratum if left untouched. Depending on the species present, test sites may have to harvest/test the EB colonies before they get too thick or 3-dimensional. EB are found as important taxa in benthic communities and reported in fouling communities from tropical to temperate regions (Watts et al. 1998; Swain et al. 2000; Mackie et al. 2012) and this ubiquitous distribution could enhance their value as hard fouling adhesion strength test taxa. The results presented herein suggest that test sites with the presence of EB could implement this methodology into routine assessments of FR coatings. One thing to note is that in certain regions bay and estuary fouling organisms are seasonal or influenced by broad scale oceanographic processes (e.g. Sorte and Stachowicz 2011; Needles and Wendt 2013), and thus they may not be consistently present for assessments of adhesion strength. For example, at the field site in California, barnacles were absent or in such low abundance as to be impractical for adhesion strength assessments for years following the marine heat wave of 2014 and El Ni~ no of 2015 2016. The addition of EB as adhesion strength assessment taxa would supplement other adhesion taxa that vary spatially or temporally. Differences in CRS were observed among barnacles, tubeworms, and EB with consistent adhesion strength patterns at both test sites (California and Florida). Similar trends were found by Kavanagh et al. (2001) who reported higher adhesion strengths of oysters and tubeworms compared to barnacles on RTV 11-based silicone FR coatings. The authors also identified differences in adhesion strength based on the coating (Kavanagh et al. 2001), as observed within the present study. This is a common occurrence reported in the fouling literature and seen in coatings testing, as different oils and other additives can alter the adhesion strength of fouling organisms (Swain et al. 2000). While EB were found to have lower adhesion values compared with the other suite of hard fouling 7 organisms, they are still a concern for the shipping industry due to their low growth form. When EB, such as Watersipora cf. subvoidea are present, they have the potential to be missed by a grooming or cleaning tool, which then removes the higher form fouling taxa such as barnacles and tubeworms, leaving open substratum for the EB to grow (Hearin et al. 2016; Hunsucker et al. in review). Additionally, EB colonies with their low form may remain attached to a ship’s hull, even when subjected to high speeds. Hunsucker et al. (2019) recently found EB to remain on a range of silicone FR coatings subjected to speeds of 10 m s 1. This may have implications for invasive species transport or fuel costs due to increased drag. EB colonies have been shown to provide an important habitat to numerous other invertebrates, potentially further exacerbating transport of non-native species to new communities if colonies remain on ships’ hulls while traveling from port to port (e.g. Sellheim et al. 2010). The accumulation of adhesion data helps to further the understanding of the interaction between EB and FR coatings, which can aid in effective hull fouling management strategies for these taxa. The adhesion mechanisms across biofouling taxa will contribute to differences in adhesion strength. The three taxa tested in this study are from a diverse array of invertebrate lineages that include a variety of substratum adhesion strategies. However, existing knowledge about these strategies often include significant gaps in the biological processes of adhesion as well as the physical properties of the adhesive. It must be acknowledged that there are few data about the adhesion mechanisms for EB and tubeworms and even less information about the relationship between adhesion mechanisms among the three taxa tested here. Previous work has shown interactions between silicone polymer coatings and barnacle adhesive due to interruptions in the physical properties (e.g. Wiegemann 2005) and the biochemistry of the adhesive related to compounds associated with several silicone polymer coatings (e.g. Rittschof et al. 2011). While these studies did not include tubeworms or EB, the results presented here, using two silicone polymer FR coatings and obtaining consistent results in adhesion strength trends across three distinct invertebrate taxa, may suggest that a similar process could be affecting the adhesive properties in tubeworms and EB. It is difficult to discuss, in depth, the potential interactions between the biochemistry of the adhesives utilized by the fouling organisms tested in this study and silicone FR coatings due to the lack of 8 G. T. WALTZ ET AL. information on the physical and biochemical properties of tubeworm and EB adhesives. However, if the process is similar for tubeworms and EB relative to a proposed barnacle adhesion model (e.g. Rittschof et al. 2011), it would be expected that an interruption in the protein bonds and enzymes associated with the adhesives for these two taxa would be seen. The results presented in this study point to variability in adhesive properties among invertebrate fouling taxa but suggest that silicone polymer coatings may affect these adhesives in similar ways. The difference in adhesion strength found here and from previous studies among diverse invertebrate taxa provides evidence that silicone based polymer coatings reduce the adhesion strength of fouling taxa and that there is variability in the adhesives utilized by these invertebrate fouling taxa (e.g. Brady and Singer 2000; Wiegemann 2005; Holm et al. 2006; Rittschof et al. 2011). Furthermore, there were differences detected in adhesion strength between two of the EB taxa tested at California on Intersleek 757. This suggests that even species in the same order and, presumably utilizing similar adhesion mechanisms, have a differential response to the same silicone polymer FR coatings. Future work examining the biochemical composition of tubeworm and EB adhesives is needed to further assess whether and how silicone polymer coatings may be impacting the functionality of these adhesives. Doing so will likely clarify how the adhesive properties between and within these taxa differ and whether silicone polymer coatings are affecting the physical properties or biochemical pathways in similar ways for barnacles, tubeworms, and EB. This would provide useful information for the development and improvement of silicone polymer coatings against multiple invertebrate fouling taxa. In addition to interspecific variability in adhesion strength to silicone polymer coatings, studies have demonstrated intraspecific variability in adhesion strength for barnacles to silicone polymer coatings (e.g. Holm et al. 2005). This suggests that, in addition to variable responses in adhesion across species, there is additional variation from the genotype within a species. The intraspecific response for wild populations of barnacles is unknown and there is no laboratory or field testing for intraspecific genotypic variability in tubeworms or EB species. Elucidating the mechanisms of heritable intraspecific differences in response to FR coatings could provide further insight in the performance, or lack thereof, of coatings developed to function as FR. This may be important when considering that many common fouling organisms have a wide global distribution and there is the potential for transport of FR resistant genotypes, quickly, on ships’ hulls or in ballast water. Spread of these more resistant genotypes could invalidate attempts to deploy previously successful FR coatings. Exposure during the testing phase of FR coatings to various ‘wild-type’ genotypes may improve the down-select process incorporated into some coating development procedures. Due to the ubiquitous distribution of tubeworms, barnacles, and EB, it is vital that FR coatings perform well against these organisms, as they are likely to be encountered in many, if not all, of the places where naval or other globally traveling ships may sail. The dynamic nature of the ocean contributes to the variability observed in the biofouling community. Successful FR coatings will have to perform well against a variety of biofouling taxa, which change spatially and temporally. Understanding the way in which different biofouling taxa respond to different coating formulations will assist with the down-select process utilized to develop new coatings (e.g. Briand 2009; Wendt 2017). Maintaining a diverse suite of static immersion field test sites, to capture as much biofouling diversity and variability as possible, will be critical to thoroughly assessing and improving novel FR coatings. Additionally, cultivating an understanding of how different coating formulations perform against different fouling taxa will continue to improve the performance of novel FR coatings particularly with respect to predicted changes in ocean conditions and biofouling communities. Conclusions This study assessed the applicability of implementing EB into the current FR testing protocol, comparing the adhesion measurements between two spatially distinct field sites. The results demonstrated that adhesion strength and growth change when coatings are exposed to differing oceanographic conditions and fouling communities. However, at both locations (Florida and California), EB were found to have a lower adhesion strength than barnacles and tubeworms. They were easy to isolate and remove using the methodology laid out in ASTM D5618. Incorporating EB into field testing, will also provide field test sites with another mode of assessing the FR properties of ships’ hull coatings, through the addition of new taxa for adhesion testing. Spatial and temporal variability in marine biofouling communities is already high and is likely to increase with global BIOFOULING climate change. FR coating developers will need performance data from across a broad suite of taxonomic categories and geographic locations in order to produce globally effective coatings. In turn, FR coatings that work on a broad spectrum of fouling organisms will likely perform better in these dynamic communities. Acknowledgements The authors thank the members of the Center for Corrosion and Biofouling Control at FIT and the DEW Lab at Cal Poly for their assistance in the field and laboratory. Lenora Brewer was an invaluable mentor and colleague. Thank you to the anonymous reviewers for feedback that greatly improved this publication. Disclosure statement No potential conflict of interest was reported by the author(s). Funding The authors would like to thank the Office of Naval Research for funding this work (award numbers N000141613208 and N000141613123). ORCID K. Z. Hunsucker http://orcid.org/0000-0003-1987-8545 G. Swain http://orcid.org/0000-0002-6580-5323 References ASTM D5618-20. 2020. 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