Exploration of Antifouling Potential of the Brown

J. Ocean Univ. China(Oceanic and Coastal Sea Research)https:///10.1007/s11802-018-3524-8ISSN 1672-5182, 2018 17 (5): 1135-1141/xbywb/E-mail:xbywb@Exploration of Antifouling Potential of the Brown AlgaeLaminaria ‘Sanhai’LI Xiangmin, LI Fengchao, JIAN Huimin , and SU RongguoKey Laboratory of Marine Chemistry Theory and Technology, Ministry of Education, Ocean University of China,Qingdao 266100, China(Received April 14, 2017; revised October 20, 2017; accepted November 13, 2017)© Ocean University of China, Science Press and Springer-Verlag GmbH Germany 2018Abstract Seaweeds are one of the largest producers of biomass in the marine environment. It has been well known that marine algae, especially brown algae was a rich source of biogenic compounds with antifouling potential that could be ideal alternatives of tributyltin (TBT). In this paper, antifouling potential of the brown algae Laminaria ‘sanhai’ was explored. Firstly, the dried alga was extracted and the antialgal and antilarval activities were investigated. The EC50 and LC50 values of crude extract of Laminaria ‘san-hai’ against diatom (Skeletonema costatum) and barnacle larval(Chthamalus challengeri) were 8.9μg mL−1 and 12.0μg mL−1 respec-tively. Then, guided by bioassay, the bioactive substances were isolated by liquid-liquid extraction. The antialgal and antilarval ac-tivities of isolated fraction were improved with the EC50 value of 7.4μg mL−1 against S. costatum and LC50 value of 9.7μg mL−1 against C. challengeri larvae. Identification by IR, Q-TOFMS and GC-MS of the isolated bioactive substances revealed the abun-dance of fatty acids. These fatty acids, most with 16, 18 or 20 carbon atoms, contained myristic, hexadecanoic, oleic, linolenic, ara-chidonic and eicosapentaenoic acids. The results indicated that both the crude extract and the isolated bioactive substances had high antialgal and antilarval activities with no highlighted cytotoxicity which made the brown algae Laminaria ‘sanhai’ a promising source of the environmentally friendly antifoulants.Key words antifouling; artificial breeding; brown algae; crude extract; fatty acids; IR Q-TOFMS and GC-MS1 IntroductionBiofouling, a natural phenomenon, causes many prob-lems to engineered structures such as ships, offshore rigs and jetties by way of increased use of manpower, fuel, material and dry-docking time (Callow et al., 2002; Chambers et al., 2006). Since the early 1960s, tributyltin (TBT) has been used widely in marine paint formulations, but evidence of harmful effects to many non-target or-ganisms was followed (Evans et al., 1995; Omae, 2003; Yebra et al., 2004). Due to the environmental pressure caused by TBT, the International Maritime Organisation (IMO) banned the use of triorganotin-based paints worldwide (Champ, 2000). In order to develop effective but environmentally friendly natural antifoulants, work is progressing worldwide (Hellio et al., 2002; Sipkema et al., 2005; Raveendran et al., 2008).Among these investigations, marine natural products have been highlighted as promising environmental friendly antifoulants (Kjelleberg and steinberg, 1994; Ye-bra et al., 2004; Raveendran et al., 2008). Many organ-isms including marine seaweeds, sponges, corals, ascid-ians and others are believed to produce rich natural anti- * Corresponding author. E-mail: jianhm@ fouling substances that could get rid of fouling organisms (Kharchenko et al., 2012). Some novel natural products for defense purposes had been isolated and investigated, and they all showed antifouling activities with less toxic and more effective compared to the organotin compounds (Proksch et al., 2002; Qian et al., 2010), which indicated the strong antifouling potential of marine natural products. For example, Seaweeds, one of the most studied marine organisms for the isolation of AF compounds, were found to produce a variety of bioactive compounds with high antifouling activity, including fatty acids, lipopetides, amides, alkaloids, terpenoids, lactones, pyrroles and ster-ols (Bhattarai et al., 2006) .The brown macroalgae, one widespread species of seaweeds, had been paid much attention for its high anti-fouling potential and some antifouling compounds were isolated and identified successfully, such as diterpene alcohols from Dictyota menstrualis (Schmitt et al., 1995, 1998), diterpenes from Canistrocarpus cervicornis (Bi-anco et al., 2009), and fatty acids from Sargassum (Hos-sain et al., 2003; Kornprobst, 2005).In this paper, the antifouling potential of the brown macroalga Laminaria ‘sanhai’ was explored. The Lami-naria ‘sanhai’, belonging to Phaeophyceae, Laminariales, Laminariaceae, Saccharina, was achieved by artificial breeding (Laminaria japonica♀ × Saccharina latissimaLI et al . / J . Ocean Univ . China (Oceanic and Coastal Sea Research) 2018 17: 1135-11411136 ♂) with excellent qualities on the basis of the original varieties such as high-temperature-resistance and high production, and it was widely planted in China and com-mercially available. We investigated the antialgal and antilarval activities of the crude extract and isolated bio-active substances from Laminaria ‘sanhai’, and identified their chemical structures by IR, Q-TOFMS and GC-MS.2 Materials and Methods2.1 Sample Pretreatment The Laminaria ‘sanhai’ was provided by Culture Col-lection of Seaweed of the Ocean University of China. The Laminaria ‘sanhai’ sample was rinsed in sterile water and 5% ethanol in order to remove associated micro flora. Cleaned samples were then surface dried by pressing it briefly between the sheets of filter papers and air dried under shade at room temperature in order to prevent photolysis and thermal degradation of metabolites. The dried seaweeds were ground to powder and packed in polyethylene bags and stored in moisture free place be-fore use. 2.2 Extraction and Purification2.2.1 The crude extractThe dried algae was suspended by stirring in 95% etha-nol (200 g in 300 mL) for 4 h at 4℃, the resultant pellet was re-extracted five times in the same way. The alco-holic extracts were combined and evaporated under vac-uum at low temperature (< 40℃). Distilled water (100 mL) and dichloromethane (400 mL) were then added, shaked well and stratified. The organic phase was collected, left dry in presence of Na 2SO 4 for 24 h, filtered and concen-trated under vacuum at low temperature until the volume reaches 5 mL to 10 mL. Following that, the concentrated extract was transferred to a 50 mL centrifuge tube and dried by blowing N 2 (Hellio et al ., 2001). Then the dried crude extract was weighted, finally the dried crude extract dissolved in 0.5 mL DMSO and diluted with Milli-Q wa-ter to obtain a 50 mg mL −1 stock solution stored in refrig-erator before use.2.2.2 Purification of the crude extractThe crude extract was applied in a C18 solid phase ex-traction (SPE) column developed with methanol andeluted with n-hexane and dichloromethane (100%n-hexane, 5:5, 100% dichloromethane) into three frac-tions. The individual fractions were screened for antialgalactivities. Based on the results of antialgal activities, thefractions that was eluted using n-hexane: dichloro-methane (5:5) and 100% dichloromethane were combined and concentrated under vacuum (< 40℃), and then solu-bilized in 20 mL CH 2Cl 2 and partitioned by NaOH (0.2 mol L −1) three times. Then the aqueous phases were com-bined, neutralized by H 2SO 4 (1.0 mol L −1) and extracted by dichloromethane. The finally organic phases resulting from this neutralization were collected and concentrated under vacuum at low temperature (< 40℃) and then dis-solved in 0.2 mL DMSO and diluted into solution (10 mg mL −1) using Milli-Q water before use. Following each step of purification procedure, the antialgal tests were performed to make sure that the bioactive substances were kept.2.3 BioassaysIn this study, diatom Skeletonema costatum (S. co-statum ) and barnacle larval Chthamalus challengeri (C.challengeri ) were used in antifouling activity tests. Dia-toms were the main components of slime films in marine environments. It attached to and reproduced on all but the most toxic surface (Marsalek et al ., 1979; Baier, 1980; Callow et al ., 1986). Barnacle larval was also a common kind of fouling organism worldwide (Swain et al ., 1998; Brady Jr et al ., 2000). They were often used as test or-ganisms for antifouling activities of antifouling com-pounds. In these experiments, we had made sure that the DMSO volume content (< 0.1%) was safe to the target fouling organism (Okumura et al ., 2001). The filteredfresh sea water (GF/F Filter, 0.45 μm) was used in thewhole experiment.2.3.1 Antialgae activities The marine diatom S. costatum was obtained from the Marine Eco-Pollution Chemistry Laboratory of the Ocean University of China. The S. costatum was cultivated in 250 mL Erlenmeyer flasks containing Guillard’s F/2 me-dium under continuous illumination (5000 lux white fluo-rescent lamps) at 20℃ with a 12 h:12 h light:dark cycle.Culture medium were all autoclaved (120℃, 20 min) andthen inoculated with 20 mL cultivated microalgae in the exponential growth phase in 5 L Erlenmeyer flasks con-taining 3 L media under aseptic conditions. The cultureswere shaken three times daily by hand to allow sufficient gas exchange until the microalgae were incubated to the exponential growth period (3 or 4 d) and reached a den-tistry of 105 cells mL −1, then they were collected at the light cycle time for the test. All the experiments were carried out in triplicate. Tests for bioactivity were performed in 250 mL Erlenmeyer flasks filled with 100 mL S. costatum suspension in ex-ponential growth period. The chlorophyll-a fluorescenceintensities (FI) were determinated by a F4500 fluorometer(Hitachi, Japan) as an indicator of algal biomass (Suggettet al ., 2011). A decrease in chlorophyll-a FI indicatedalgae growth inhibition by the extract. The percentage ofgrowth inhibition was calculated as:% inhibition = (FI C – FI T )/ FI C *100,where FI C was the mean chlorophyll-a fluorescence in-tensity of the controls and FI T was the mean chloro-phyll-a fluorescence intensity of the test samples. 2.3.2Antilarval activities The Chthamalus challengeri were collected from the in-tertidal zone in Zhanqiao, Qingdao. After collected,LI et al . / J . Ocean Univ . China (Oceanic and Coastal Sea Research) 2018 17: 1135-11411137washed the sludge and other organisms away and dried in shade, the Chthamalus challengeri was cultured with fresh filtered seawater. Under light irradiation, the adult barnacle released larvae. The larvae were collected, cul-tured for 24 h, and then could be used in the antilarval experiments.The anti-larval experiments were conducted in sterile 3 mL 24-well polystyrene plates. The filtered seawater (0.45 μm) was used as control. Each well contained about 30 barnacle larvae in 3 mL of the test solution. The plates were incubated at 25℃ for 24 h in plant growth chamber under in the dark. All tests were performed in triplicate. The antilarval activities were determined by counting the dead and live individuals and expressing the result as a proportion of the total number of larvae in the well (the percentage of larval death).2.4 Statistical AnalysisThe data from antialgal and antilarval tests were ana-lyzed by SPSS Version 11.5 software package. The dif-ferences between the experimental treatments and the control samples were determined using one-way ANOV As followed by Dunnett’s test at 95% confidence level. The EC 50s were calculated as the concentration at 50% of the maximal effect of S. costatum compared with the control and the LC 50s as the concentration at which 50% of larvae were dead compared with the control. And they were computed from the test results using probit analysis.2.5 Identification of the Isolated Bioactive SubstancesThe isolated bioactive compounds were analyzed and identified by IR, Bruker maXis quadrupole time-of-flight mass spectrometry (Q-TOFMS) and ITQ-1100 gas chro-matography-mass spectrometry (GC-MS).3 Results and Discussion3.1 Antialgal and Antilarval Activities of the CrudextractFor algal inhibiting test, the proper volumes of the crude extract stock solution were added to algae cultures so that its concentrations were 0, 5.0, 7.5, 10.0, 15.0, and 25.0 μg mL −1, respectively, and the chlorophyll-a FI were recorded every 24 h for 96 h. Based on ANOV A and Dun-nett’s analyses, the crude extract, when tested at 7.5 μg mL −1, displayed significant toxicity when compared to the control (P < 0.05). The crude extract with concentrations below 7.5 μg mL −1 did not inhibit the growth of S. co-statum . But when the crude extract concentration was increased up to 10.0 μg mL −1, the algal growth was sig-nificantly inhibited (Fig.1). The EC 50 of the crude extract against S. costatum was 8.9 μg mL −1.A series of aqueous solutions were prepared for anti-larval activity test by diluting the crude extract stock so-lution with the filtered fresh sea water, and the concentra-tions were set at 0, 5.0, 7.5, 10.0, 20.0 and 30.0 μg mL −1,respectively. After 24 h exposure, the crude extract in-duced C. challengeri larvae death in a dose-dependent manner (Fig.2). Based on ANOV A and Dunnett’s analyses, the crude extract, when tested at 7.5 μg mL −1, displayed significant toxicity when compared to the control (P < 0.05). When the concentration of the crude extract was above 20.0 μg mL −1, more than half of test barnacle larvae were killed and the LC 50 of the crude extract against C. challengeri larvae was 12.0 μg mL −1.Fig.1 Growth curve of S. costatum subjected to different concentrations of the crude extract during a 96 h growthperiod.Fig.2 Lethal effects of the crude extract against C . chal-lengeri larvae after 24 h exposure.Hellio et al . (2002) reported that the dichloromethane extract of Ulva lactuca could inhibit the growth of the diatom Phaeodactylum tricornutum with EC 50 of above 300.0 μg mL −1. They (Hellio et al ., 2001) also found that the crude extract of brown algae Bifurcaria brassicae-formis had antialgal activity and when its concentration was up to 30 μg mL −1, more than 60% attachment of spores of macroalgae (such as Enteromorpha intestinalis ) was inhibited. Águila- Ramírez et al . (2012) presented that the MIC (the minimum inhibitory concentrations) values of crude extracts of Ulva lactuca and Laurencia johnstonii against some species of temperate marine mi-croalgae were ranged between 10 and 25 μg mL −1. The antifouling activities of the dichloromethane extract of Himanthalia elongata and crude extract of Lyngbay ma-LI et al . / J . Ocean Univ . China (Oceanic and Coastal Sea Research) 2018 17: 1135-11411138 juscula were also reported with with an LC 50 of 96.2 μg mL −1 against Amphibalanus amphitrite (Maréchal and Hellio, 2011) and an LC 50 of 20 μg mL −1 against Amphi-balanus amphitrite , respectively (Tan et al ., 2010). Comparatively, the crude extract of Laminaria ‘sanhai’ had significantly higher antialgal and antilarval activities.3.2 Antialgal and Antilarval Activities of thePurified Bioactive SubstancesThe stock solution of purified substances was added toalgae cultures to make its concentrations 0, 2.5, 5.0, 7.5,10.0, and 20.0 μg mL −1, respectively, and the chloro-phyll-a FI were recorded every 24 h for 96 h (Fig.3). Based on ANOV A and Dunnett’s analyses, the purified substances, when tested at 7.5 μg mL −1, displayed signifi-cant toxicity when compared to the control (P < 0.05). The purified substances at concentration below 5.0 μg mL −1 had no obvious algal growth inhibition and when the concentration of the purified substances reached upto10.0 μg mL −1, the FI of S. costatum was too weak to be detected. The EC 50 of purified substances toward S. co-statum was determinated as 7.4 μg mL −1. Aqueous solutions of 0, 5.0, 7.5, 15.0, 25.0 and 30.0 μgmL−1 purified substances were prepared for antilarval activity test. Exposure to the purified substances for 24 hFig.3 Growth curve of S.costatum subjected to different concentrations of purified substances during a 96 h growth period.Fig.4 Lethal effects of the purified substances against C . challengeri larvae after 24 h exposure.also increased the C. challengeri larvae death in a dose- dependent manner (Fig.4). Based on ANOV A and Dun-nett’s analyses, the purified substances, when tested at 7.5 μg mL −1, displayed significant toxicity when compared to the control (P < 0.05). More than half of the larvae were dead at the concentration of 15.0 μg mL −1 compared to the control. And when the concentration of the purified sub-stance reached up to 25.0 μg mL −1, all of the tested larvae died. The LC 50 of purified substances toward C. chal-lengeri larvae was 9.7 μg mL −1. Some researchers had reported some natural bioactive compounds with antifouling potential. The palmitic acids from macroalgae Sargassum muticum could inhibit thegrowth of the diatom Cylindrotheca closterium with an EC 50 value of 45.5 μg mL −1(Bazes et al ., 2009). Two fatty acids, 9, 12-Octadecadienoic acid) and n-Hexadecanoicacid, isolated from Laurencia brandenii , showed antilar-val acitivities with the EC 50 of 0.912 mg mL −1and LD 50 of 1.48 mg mL −1against Balanus amphitrite settlement, re-spectively (Manilal et al ., 2010), the bis-1-oxaquinolizi-dine alkaloids isolated from marine sponge Haliclona exigua showed settlement inhibition activity against bar-nacle cypids with LC 50 of 18.07 μg mL −1(Raveendran et al ., 2008), and the linear diterpenes from brown algae Bifurcaria bifurcate had a LC 50 value of 38.3 μg mL −1against cyrids of Balanus amphitrite (Maréchal et al ., 2004). Recently, Cho reported that the chromanol class isolated from brown algae Sargassum horneri with EC 50 of 0.08-1.79 μg mL −1 against diatom Navicula annexa and EC 50 of 1.4-6.4 μg mL −1 agaist mussel larvae settlement (Cho, 2013). From the above, the purified bioactive sub-stances from Laminaria ‘sanhai’ had high antialgal and antilarval activities and could be considered as a promis-ing source of natural antifoulingcompounds.3.3 Identification of the Bioactive CompoundsThe bioactive substances with high antialgal and anti-larval activities was isolated through the bioassay-guided isolation procedure, in which, the alkali washing and ex-traction under acidic conditions were key steps that indi-cated that the bioactive substances should be fatty acid compounds. After bioassays, the isolated bioactive sub-stances were subjected to IR, Q-TOFMS and GC-MSFig.5 IR analysis of the bioactive compounds.LI et al . / J . Ocean Univ . China (Oceanic and Coastal Sea Research) 2018 17: 1135-11411139analysis for chemical structure elucidation.3.3.1 IR identification of the bioactive compounds The IR analysis result was shown in Fig.5. There were three strong peaks. Two of the peaks’ wave-numbers were about 3000 which was in accord with the IR wave number of the carboxylic acid hydroxyl (3400 2500 cm −1).The wave number of about 1700 was the IR characteristic peak of carbonyl group in carboxylic acid. So the IR de-tection confirmed the existence of carboxyl group and the isolated bioactive substances should be mainly composed of fatty acid compounds.3.3.2 Q-TOFMS identification of the bioactivecompoundsNegative mode ionization that removed a hydrogen ion was developed to screen for possible candidate com-pounds (Fig.6). SmartFormula3D was used to identify the fragment and give the sum formula information in the analysis. The bioactive compounds contained 16, 18 or 20carbon atoms based on the molecular formula (Table 1). So the isolated bioactive compounds should be a series of unsaturated and saturated fatty acids that contains 16 or 18 carbon atoms based on the molecular formula pro-vided by Q-TOFMS and the functional group provided by the IR. The exact chemical structures of the isolated bio-active compounds would be given by the following GC- MS analysis.Table 1 the molecular formulae, average mass accuracies of the observed fragmentsQ-TOFMSEntry[M-H] −Molecular formula1 227.1966 C 14H 27O 22 255.2265 C 16H 31O 23 277.2103 C 18H 29O 24 279.2260 C 18H 31O 25 281.2416 C 18H 33O 2 6 283.2567 C 18H 35O 2 7 301.2100 C 20H 29O 28 303.2255C 20H 31O2Fig.6 Negative mode ionization of the bioactive compounds provided by the Q-TOFMS.3.3.3 GC-MS identification of the bioactive compounds Results from the optimized GC-MS analysis indicated the main active ingredients of the isolated bioactive sub-stances were ten fatty acids, oxalic acid, myristic acid,hexadecanoic acid, octadecanoic acid, 9-octadecenoic acid, 9,12-octadecadienoic acid, 6,9,12-octadecatrienoic acid, 9,12,15-octadecatrienoic acid, 6,9,12,15-Eicosatet-raenoic acid, 5,8,11,14-eicosatetraenoic acid and cis-5, 8, 11, 14, 17-eicosapentaenoic acid (Fig.7), they were a se-ries of unsaturated and saturated 16, 18 or 20 carbon fatty acids. Among those fatty acid compounds, Octadecanoicacid accounts for 21.64% by GC peak area integrationwith total area detected normalized to 100%, hexa-decanoic acid for 20.11%, oxalic acid for 18.49%, oleicacids for 8.86%, and myristic acid for 6.29% (Table 2). Some fatty acids could exert a high level of inhibition against certain species of microorganism (Willett and Morse, 1966; Miller et al ., 1977) and people have attach-ed much importance to fatty acids as a promising alterna-tive to TBT (Russel, 1991; Bhattarai et al ., 2006). Hexa-decanoic acid, isolated from macroalgae Sargassum mu-ticum , showed good antialgal activity against the diatomTable 2 The compounds identified by GC-MS analysisEntryTime (min)Name of the compoundPeak area (%)1 6.487 Myristic acid 6.292 7.801 Hexadecanoic acid 20.113 9.010 Octadecanoic acid 21.644 9.132 9-Octadecenoic acid 8.865 9.396 9,12-Octadecadienoic acid 4.32 6 9.583 6,9,12-Octadecatrienoic acid 1.57 7 9.791 9,12,15-Octadecatrienoic acid 2.808 10.007 6,9,12,15-Eicosatetraenoic acid 4.989 11.182 5,8,11,14-Eicosatetraenoic acid 3.58 10 11.802 cis-5,8,11,14,17-Eicosapentaenoic acid 2.99LI et al . / J . Ocean Univ . China (Oceanic and Coastal Sea Research) 2018 17: 1135-11411140 Cylindrotheca closterium with an EC 50 value of 45.5 μg mL −1 (Bazes et al ., 2009). Octadecanoic acid, 5,8,11,14- Eicosatetraenoic acid, and 5,8,11,14,17-Eicosapen-tae- noic acid, isolated from two congeneric sponges from Hong Kong and the Bahamas, had been proved with highly antibacterial activities (Lee et al ., 2007). Suber-gorgic acid, produced by a gorgonian, inhibited settle-ment of Balanus amphitrite larvae with LC 50 value of 200 μg mL −1 (Qi et al ., 2008). Myristic and hexadecanoic ac-ids, isolated from marine fungus Aureobasidium pullulans , showed high inhibition effects against Skeletonema co-statum and Amphibalanus amphitrite larval with an EC 50 of 49.4 μg mL −1 and LC 50 of 22.2 μgmL −1 respectively (Gao et al ., 2013). Comparatively, the fatty acid com-pounds isolated from Laminaria ‘sanhai’ exerted much higher antifouling activity against S. costatum and C. challengeri .Fig.7 GC-MS analysis of the bioactive compounds.4 ConclusionsIn conclusions, the bioactive substances isolated from artificial breeding Laminaria ‘sanhai’ had high antialgaland antilarval activities, and eleven fatty acid compounds were identified as the main active ingredients . Unlike thehigh toxicity and environmental persistency of the or-ganotin compounds, those natural bioactive compounds are reversible toxicity and non-residual effect, whichmade them promising sources of environmental friendly antifoulants. Acknowledgements This study was supported by the National Natural Sci-ence Foundation of China (No. 41376106), the NationalKey Research and Development Program (No. 2016YFC1402101) and the research program from Na-tional Marine Hazard Mitigation Service (No.2014AA060). We are thankful to Prof. Tao Liu of Col-lege of Marine Life of Ocean University of China for providing the Laminaria ‘sanhai’. References Águila-Ramírez, R. N., Arenas-González, A., Hernández-Guerrero, C. J., González-Acosta, B., Borges-Souza, J. M.,Véron, B., Pope, J., and Hellio, C., 2012. Antimicrobial and antifouling activities achieved by extracts of seaweeds from Gulf of California, Mexico actividades antimicrobianay anti- incrustante obtenidas de los extractos de algas marinas del Golfo de California, México. Hidrobiológica , 22 (1): 8-15. Baier, R. E., 1980. Substrata influences on adhesion of micro-organisms and their resultant new surface properties. In: Ad-sorption of Microorganisms to Surfaces . Bitton, G ., and Mar-shall, G ., eds., Wiley, New York, 59-104.Barbosa, J. P., Fleury, B. G ., da Gama, B. A. P., Teixeira, V . L., and Pereira, R. C., 2007. Natural products as antifoulants in the Brazilian brown alga Dictyota pfaffii (Phaeophyta, Dic-tyotales). Biochemical Systematics and Ecology , 35 (8): 549- 553.Bazes, A., Silkina, A., Douzenel, P., Fa, F., Kervarec, N., Morin, D., Berge, J. P., and Bourgougnon, N., 2009. Investigation of the antifouling constituents from the brown alga Sargassum muticum (Yendo) Fensholt. Journal of Applied Phycology , 21 (4): 395-403.Bhadury, P., and Wright, P. C., 2004. Exploitation of marine algae: Biogenic compounds for potential antifouling applica-tions. Planta , 219 (4): 561-578.Bhattarai, H. D., Lee, Y . K., Cho, K. H., Lee, H. K., and Shin, H. W., 2006. The study of antagonistic interactions among pe-lagic bacteria: A promising way to coin environmental friendly antifouling compounds. Hydrobiologia , 568 (1): 417- 423.Bianco, E. M., Teixeira, V . L., Pereira, R. C., de Souza, A. M., Nucci, P., Afonso, I. F., Rodrigues, C. R., and Castro, H. C., 2009. Brown seaweed defensive chemicals: A structure-ac- tivity relationship approach for the marine environment.Natural Product Communications , 4 (2): 173-178.Brady Jr., R. F., and Singer, I. L., 2000. Mechanical factors fa-voring release from fouling release coatings. Biofouling , 15 (1-3): 73-81.Callow, M. E., 1986. Fouling algae from ‘in-service’ ships. Botanica Marina , 24 (4): 351-358.Callow, M. E., and Callow, J. A., 2002. Marine biofouling: Asticky problem. Biologist , 49 (1): 1-5. Chambers, L. D., Stokes, K. R., Walsh, F. C., and Wood, R. J.,2006. Modern approaches to marine antifouling coatings. Surface and Coatings Technology , 201 (6): 3642-3652. Champ, M. A., 2000. A review of organotin regulatory strategies, pending actions, related costs and benefits. Science of the To-tal Environment , 258 (1): 21-71.Cho, J. Y ., 2013. Antifouling chromanols isolated from brown alga Sargassum horneri . Journal of Applied Phycology , 25 (1):299-309.Culioli, G ., Ortalo-Magné, A., Valls, R., Hellio, C., Clare, A. S.,and Piovetti, L., 2008. Antifouling activity of meroditerpe-noids from the marine brown alga Halidrys siliquosa . Journal of Natural Products , 71 (7): 1121-1126. Evans, S. M., Leksono, T., and McKinnell, P. D., 1995. Tribu-tyltin pollution: Adiminishing problem following legislation limiting the use of TBT-based anti-fouling paints. MarinePollution Bulletin , 30 (1): 14-21.Galbraith, H., and Miller, T. B., 1973. Physiological effects oflong chain fatty acids on bacterial cells and their protoplasts. Journal of Applied Bacteriology , 36 (4): 647-658.Hellio, C., De La Broise, D., Dufosse´, L., Le Gal, Y ., and Bourgougnon, N., 2001a. Inhibition of marine bacteria by ex-tracts of macroalgae: Potential use for environmentallyfriendly antifouling paints. Marine Environmental Research , 52 (3): 231-247.。