ReseaRch PaPeR Journal of Agricultural and Marine Sciences Vol. 20 (2015): 30-33. Reveived 5 Aug. 2014 Accepted 19 Feb 2015 Microfouling on biocidal and non-biocidal antifouling coatings Thirumahal Muthukrishnan1* and Sergey Dobretsov 1 *1 Thirumahal Muthukrishnan ( ) Sultan Qaboos University, Col- lege of Agricultural and Marine Sciences, Department of Marine Sci- ence and Fisheries. Box 34, Al-Khod 123. Sultanate of Oman. email: mthirumahal@hotmail.com Introduction Biofouling has been a major problem in several ma-rine industries including the shipping industry. Hull fouling adversely affects hydrodynamic drag الرتاكم احليوي للكائنات الدقيقة على الطالءات القاتلة وغري القاتلة املضادة للرتاكم على األسطح املغمورة ثريوحمال موثوكرشنان1* وسرجي دوبريتسوف 1 Abstract. Although antifouling marine paints have been used to prevent biofouling, not much is known about their effectiveness in preventing attachment of microorganisms. The current study aims at estimating the abundance of bacteria within biofilms developed on various commercial antifouling coatings in Marina Bandar Rowdha and Ma- rina Shangri La, Oman. Coatings tested included Pettit #1863 and #1792, West Marine #11046620, #5566252 and #10175206, Hempel Hard Racing #76484, Hempel Olympic #86950, Hempasil X3 and International YBA920. All coat- ings were applied on clean plastic slides. Slides without any coating were used as controls. Microbial biofilms were harvested after 2, 7 and 14 days of biofouling. Bacterial density was estimated using epifluorescence microscopy. There was a significant difference between the various treatments (coatings and control) after 2, 7 and 14 days of biofouling. Although there were significant differences between both locations after 2 and 14 days of biofouling, no significant dif- ference was observed after 7 days of biofouling at both locations. At Shangri La, the lowest bacterial density was found on International YBA920, Pettit #1792 and Hempasil X3 after 2 days, 7 days and 14 days respectively in comparison to the control treatments. However at Bandar Rowdha, International YBA920 showed the lowest bacterial density after 2 days while West Marine #10175206 showed the lowest bacterial density after both 7 days and 14 days of biofouling in comparison to the control treatment. The differential performance of tested antifouling coatings may be attributed to several factors including varying environmental conditions, difference in microfouling communities, time of exposure and physical and chemical properties of antifouling coating. Keywords: Coatings, antifouling, microfouling, bacteria, phototrophic. املستخلص: بالرغم من أن األصباغ البحرية املضادة للرتاكم على األسطح املغمورة تستخدم للحد من الرتاكم احليوي ، إال أنه ال يعرف الكثري عن مــدى كفاءهتــا يف احلــد مــن تعلــق الكائنــات الدقيقــة. وهتــدف الدراســة احلاليــة إىل تقديــر تواجــد البكرتيــا يف أغشــية حيويــة رقيقــة تكونــت علــى طــاءات جتاريــة خمتلفــة مضــادة للرتاكــم علــى األســطح يف مارينــا بنــدر الروضــة ومارينــا شــاجنري ال بســلطنة عمــان. الطــاءات ايل مت اختبارهــا مشلــت: 1863 ، #76484 Hempel hard Racing ، 10175206# و 5566252 #و West Marine #11046620 ، 1792 #و Pettit # International YBA920 ، Hempasil X3 ، #86950 Hempel Olympic. مجيــع الطــاءات مت وضعهــا علــى شــرائح باســتيكية نظيفــة مــع اإلبقــاء علــى بعــض الشــرائح غــري مطليــة الســتخدامها كمعاملــة مرجعيــة. ومــن مث مت مجــع األغشــية احليويــة الرقيقــة املتكونــة بعــد 2 و 7 و 14 يومــا مــن بدايــة الرتاكــم احليــوي علــى األســطح. ومت تقديــر كثافــة البكرتيــا باســتخدام تقنيــة اجملهــر الفــوق فلورســي. أظهــرت النتائــج وجــود فروقــات معنويــة كبــرية بــن املعامــات املختلفــة ) املطليــة وغــري املطليــة( بعــد 2 و 7 و 14 يومــا مــن بدايــة الرتاكــم احليــوي علــى األســطح .وبالرغــم مــن االختــاف املعنــوي يف النتائــج بــن املوقعــن بعــد 2 و 14 يومــا مــن الرتاكــم احليــوي علــى األســطح ، إال أنــه مل يكــن هنــاك فــرق يذكــر بعــد 7 ايــام مــن الرتاكــم احليــوي يف املوقعــن. ويف مارينــا شــاجنري ال، كانــت أقــل كثافــة للبكرتيــا يف املعامــات: International YBA920 و Pettit1792# و Hempasil X3 باملقارنــة مــع املعامــات الغــري مطليــة، بعــد 2 و 7 و 14 يومــا علــى التــوايل. أمــا يف مارينــا بنــدر الروضــة فقــد أظهــر الطــاء International YBA920 أقــل كثافــة للبكرتيــا بعــد يومــن مــن بدايــة الرتاكــم احليــوي علــى األســطح. بينمــا أظهــر الطــاء West Marine #10175206 أقــل كثافــة للبكرتيــا بعــد7 و 14 يومــا مقارنــة باملعامــات الغــري مطليــة. إن األداء املتبايــن للطــاءات املضــادة للرتاكــم احليــوي الــي مت اختبارهــا ميكــن أن ينســب إىل عــدة عوامــل منهــا الظــروف البيئيــة املختلفــة، واختــاف جمتمعــات املرتاكمــات احليويــة الدقيقــة، ومــدة التعــرض، واخلصائــص الكيميائيــة والفيزيائيــة للطــاءات املضــادة للرتاكــم علــى األســطح. الكلمات املفتاحية: الطاءات ، مضادات الرتاكم احليوي على األسطح ، الرتاكم احليوي للكائنات الدقيقة، البكرتيا، اإلجنذاب للضوء leading to elevated fuel consumption and higher main- tenance costs although the impact of biofilms is signifi- cantly less than that of macrofouling (Yebra et al. 2004; Schultz 2007; Schultz et al. 2011). In marine environ- ments, formation of biofilms (ie microfouling) depends on the types of fouling microorganisms, environmental factors such as current, temperature, salinity, nutrient levels and hydrodynamic conditions (Wieczorek and Todd 1998 ; Lau et al. 2005; Zhang et al. 2011) and prop- erties of substratum (Whitehead and Verran 2008). Mi- 31Research Article Muthukrishnan, Dobretsov crobial fouling communities consist mainly of numerous species of bacteria and diatoms that can positively and/ or negatively interact with each other (Railkin 2003; Dobretsov 2010). Both bacteria and diatoms may also have a significant impact on the recruitment of inver- tebrate larvae and algal spores (macrofouling) by either enhancing or inhibiting their settlement (Mitchell and Maki 1988; Maki 2002; Huang and Hadfield 2003; Qian et al. 2007; Hadfield 2011). This significantly influenc- es the extent to which biofouling occurs in the marine environment. However bacteria have generally been ac- cepted to be the primary colonizers on man-made sur- faces in the marine environment (Molino et al. 2009b). Therefore it is important to study the efficiency of anti- fouling coatings in preventing bacterial fouling during the primary stages of biofouling in the marine environ- ment. The objective of the current study was to estimate the abundance of bacteria within biofilms developed on various commercial antifouling coatings at two different locations in Oman. The hypothesis tested was that treat- ments (nine commercial antifouling coatings) and loca- tion influence the abundance of bacteria within biofilms developed on commercial antifouling coatings. Materials and methods Coatings preparation Six commercial antifouling coatings (Petit # 1863, Petit 1792, West Marine #5566252, West Marine #11046620, West Marine #10175206 and International Micron Ex- tra YBA 920) were obtained at local boat shop (Muscat, Oman). Three commercial antifouling coatings (Hempel Hard Racing 76484-51170, Hempel Olympic 86950- 5110 and Hempasil X3) were obtained from Hempel Ltd. Co. (Muscat, Oman). The nine antifouling coatings (Ta- ble 1) were manually applied onto cleaned, acrylic plas- tic slides (75 x 25 mm) at Marine Science and Fisheries Laboratory, Sultan Qaboos University, Oman. All coat- ed slides were dried for several hours at ambient tem- perature prior to deployment. Uncoated cleaned plastic slides were considered to be the control treatments. For each treatment including control, a total of 18 replicate slides were prepared. Coatings Deployment A total of 180 slides were randomly inserted into 6 slide cassettes (each 21 x 16 x 3 cm) such that each slide cas- sette contained 3 replicates of each treatment and 30 equally spaced slides in total. Each slide cassette was deployed by ropes such that each slide in the slide cas- sette was kept vertical with respect to the surface of seawater. Three slide cassettes were deployed each at Marina Shangri La (Muscat, Oman 23º 32’ 55”  N 58º 39’ 23” E) and Marina Bandar Rowdha (Muscat, Oman 23º 34’ 55” N 58º 36’ 27” E). Sample collection Each of the three slide cassettes at Marina Bandar Row- dha and Marina Shangri La were withdrawn after 2 days, 7 days and 14 days of biofouling respectively. During sample collection, all slides from the slide cassette were carefully transferred into clean plastic boxes containing formalin (3.7% final concentration) and immediately transferred to the laboratory at 4ºC for further analysis (see below). Estimating abundance of bacteria The total bacterial density on the treatment surfac- es was estimated by staining an area of 2 x 2 cm with 10-12  µl of 4, 6-diamidino-2-phenylindole (DAPI, Sig- ma, Germany) solution for 15 minutes according to Do- bretsov and Thomason (2011). The number of bacteria in 10 randomly selected fields of view on the ocular grid (0.001 mm2) was counted using an epifluorescence mi- croscope (Axiostar plus, Zeiss, Germany; magnification 1000x; λEx=359nm, λEm=441nm). Statistical analysis Factorial ANOVA was used to test the effect of treat- ment and location on the total bacterial density using Statistica 11 (Statsoft, USA) after 2, 7 and 14 days of bio- fouling. Post hoc HSD test was used to test for significant differences among the treatments and locations. In all cases, the threshold for significance was 0.05. Results The treatments (antifouling coatings and control) sig- nificantly influenced the bacterial density in biofilms developed after 2, 7 and 14 days of biofouling (Figure 1A and Figure 1B; ANOVA, HSD, P < 0.0001). Although Figure 1. Bacterial density in biofilms developed on all treatments (Coatings 1-9 and Control) after 2, 7 and 14 days of biofouling at (A) Marina Shangri La and (B) Mari- na Bandar Rowdha . Data are the means + SD (n=3). 32 SQU Journal of Agricultural and Marine Sciences, 2015, Volume 19, Issue 1 Microfouling on biocidal and non-biocidal antifouling coatings both locations were found to significantly affect bac- terial density after 2 and 14 days (ANOVA, HSD, P < 0.0001) there was no significant difference between both locations after 7 days of biofouling (ANOVA, HSD, P = 0.237). However both treatments and locations togeth- er significantly affected the bacterial density in biofilms after 2, 7 and 14 days of biofouling (ANOVA, HSD, P < 0.01). At Shangri La, the lowest bacterial density was found on International YBA920, Pettit #1792 and Hempasil X3 after 2 days, 7 days and 14 days respec- tively in comparison to the control treatments (Figure 1A). However at Bandar Rowdha, International YBA920 showed the lowest bacterial density after 2 days while West Marine #10175206 showed the lowest bacterial density after both 7 days and 14 days of biofouling in comparison to the control treatment (Figure 1B). The differential performance of tested antifouling coatings may be attributed to several factors including varying environmental conditions and differences in the abun- dance of fouling bacterial communities. The variation in the concentrations of biocides in these coatings may be additional factor in influencing bacterial attachment on coatings. In particular the polishing rate behavior and biocide delivery rate behavior is known to vary for dif- ferent coating types (Finnie & Williams 2010, Bressy et al. 2010). Clearly further investigations are required to study the abundance and composition of bacterial foul- ing communities on antifouling coatings. Conclusions The current study shows that the abundance of bacte- ria in biofilms developed on commercial antifouling coatings is significantly influenced by the coating types and both coatings and location together after 2, 7 and 14 days of biofouling. Varying environments were not found to affect the bacterial density after 7 days of bio- fouling although there were significant differences after 2 and 14 days of biofouling. Acknowledgements This study was supported by HM Sultan Qaboos Re- search Trust Fund SR/AGR/FISH/10/01. The authors thank Hempel Ltd. Co. (Muscat, Oman) for providing coatings. The authors would also like to thank Ms. An- nika Vaksmaa (Sultan Qaboos University) for her assis- tance in experimental setup. References Bressy, C., C., Hellio, J.P., Marechal, B., Tanguy, and A., Margaillan. 2010. 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