Antifouling strategy for marine transport efficiency in Nigeria : The isothiazolone option

The sustainability of Nigerian maritime industry is threatened by drag and deterioration caused by biofilms on painted marine structures. The bacterial population of scrapings from twenty boat hulls on Ojo waterside shores, Lagos, Nigeria and the antibacterial activity of in-can isothiazolone biocide against the isolated biofilm bacteria were investigated. The disc diffusion technique using Mueller Hinton agar (MHA) media and sterile filter paper discs immersed into the biocides was employed. Bacterial population -5 density of the scrapings ranged from 1.3 to 2.2 x 10 CFU/g. They were identified as Pseudomonas aeruginosa, Proteus mirabilis, and Staphylococcus aureus using the analytical profile index (API). All the isolates were susceptible to isothiazolone biocide with the highest inhibition zone of 24.75mm (P. mirabilis) and the lowest inhibition zone of 4.63mm (S. aureus). The MIC was observed as 15μl/ml (S. aureus), 5μl/ml (P. aeruginosa) and 5μl/ml (P. mirabilis) while MBC was 20μl/ml (S. aureus), 5 μl/ml (P. aeruginosa) and 15μl/ml (P. mirabilis). Results showed that isothiazolone biocide has broad spectrum antibacterial activity against bacterial biofilms on marine structures.


Introduction
and cost-effective running of marine transport.
Maritime transport play significant role in Much attention has been given to the provision of Nigeria's economy.In Nigeria, maritime equipment and implementation of tariff transportation of goods and services facilitates structure, manual examination of goods, trade and commerce, recreation, employment transport logistics, ports congestion, custom and job creation, revenue generation, tourism, clearance for containers and security of cargo in industrial growth and development, defense and transit (Igbokwe, 2001; Ogunsanya and security, improved international relations as well Olawepo, 2008;Ogunsiji and Ogunsiji, 2010; as socio-political harmony (Igbokwe, 2001).Ogbonnaya et al., 2011;Eniola et al., 2014).
However, according to (Lazarus and Ukpere, However, there is paucity of information on the 2011), Nigeria loses as much as $4 billion US resilient activities of microorganisms in depleting dollars to foreign ship owners yearly owing to the economic potentials of the maritime industry lack of indigenous capacity in the local maritime by slowing down boat and ship movement.The transportation.Unknown to many, biofouling Nigerian ports system has been unproductive, contributes to this loss by preventing the smooth static and malfunctioning leading to ports congestion and disruption of economic activities.
ultimately lead to growth inhibition, lack of cell Over time, the accumulation of organisms that division and irreparable damage (Nwachukwu form biofouling on hulls have caused decrease in and Akpata, 2003).Although, isothiazolone the hydrodynamic performance of vessels and biocides have been used in the UK, USA and the frictional effects has led to increased drag of other countries, there is paucity of information up to 60% resulting in 10% reduction in speed on its use as antifouling agent on boats in the (Yebra, 2004).An estimate by Liu et al. (1997) shores of Nigeria.It is therefore, the aim of this indicates that on average, fuel consumption study to evaluate and report its efficacy in increases by 6% for every 100 µm increase in the reducing antifouling of boats at the Ojo water average hull roughness caused by fouling side in Lagos, Nigeria.This is particularly organisms.
necessary because biofouling contributes to Boat hulls are susceptible to micro and making Nigerian ports user-unfriendly and macro fouling irrespective of the material from unattractive to some liners, shippers and which they are constructed.The fouling, if importers who now prefer berthing at allowed to continue proliferating, causes neighboring ports especially Cotonou port to increased drag on the hull, leading to increased berthing in Nigerian ports (Igbokwe, 2001).This fuel consumption, and can eventually cause is expected because in Nigeria, cargo throughput significant damage to the boat structure has a turnaround time of 5.25 days on the depending on the extent of infestation average (Eniola et al., 2014) which is still on the (Darbyson et al., 2009).A heavily fouled vessel high side compared to the International Maritime may also lose maneuverability.It is therefore, Organization's stipulation of 48 h.The necessary to apply some form of coating which prevention of marine biofouling on our maritime will protect the hull against infestation.These infrastructure using antifouling paints fortified coatings are generally known as antifouling with isothiazolone biocides could help achieve paints and they are applied to the hull at regular this feat.intervals because they wash off with time (Briane et al., 2013).They are also applied to

Materials and methods reduce the risk of spreading of invasive species
Sampling Locations and Procedures (Ashton et al., 2006).Studies have been Scrapings from painted boat hulls were performed investigating the ability of capsaicin aseptically collected in duplicates with a scalpel and zosteric acid as paint additives to prevent th on the 10 of April, 2015 from twenty speed biofouling (Xu et al., 2005).There have also been boats at Ojo water side, Lagos after issued reports of nanoparticle-based coatings to permission from the water side authorities.The prevent fouling (Anyaogu et al., 2008).All these 2 scraping pieces of about 1-5 cm which were have their various drawbacks.Isothiazolone firmly attached or partly detached were collected biocides on the other hand, are safe and have to a depth of about 1-1.5 mm.They were been widely used in industrial water treatment subsequently placed in sterile, properly labeled applications to control microbial growth and petri dishes which were carefully sealed and biofouling (Williams, 2007).Isothiazolone taken to the laboratory for analyses.biocides such as Kathon 5287 have been found to be highly effective, broad-spectrum and licensed for use as active antifouling agents in Microbial Isolation and Preparation of Inoculum the UK and other countries.They were the first Bacteria were isolated from the paint organic booster biocide to be registered for use scrapings in the laboratory using spread plate by the USAEPA (Voulvoulis, 2006).The killing technique (Nwachukwu and Akpata, 2003).mechanism of isothiazolone biocides include Following maceration and serial dilution, aliquots inhibition of dehydrogenase enzymes such as The plates were incubated aerobically at 37°C dehydrogenase and alcohol dehydrogenase for 24 h.After incubation, developed colonies (Chapman and Diehl, 1995).Isothiazolones were counted, purified by subculturing and have also been shown to inhibit ATP, which will preserved on NA slants at 4°C.A loop full of pure on the National Committee for Clinical overnight cultures from the labeled stock culture Laboratory Standards (NCCLS) in test tubes.The bottles was picked and introduced into sterile tubes were supplemented with varying o peptone water and incubated at 37 C for 24 h to concentrations of the biocide (5,10,15,20,40, obtain growth.Inoculum was then standardized 60, 80 and 100µl/ml).The well labeled tubes o by diluting bacterial suspension in each test tube were incubated at 37 C for 24 h.At the end of with sterile peptone water to match the turbidity incubation period, the tubes that showed no 8 of 0.5 McFarland standard (10 CFU/ml).visible microbial growth were recorded as the MIC of the biocide against the particular tested isolates (Qi et al., 2004).Subsequently, aliquots Antibacterial Activity of Isothiazolone Biocide (0.1 ml) of cell suspension from the test tubes Preparation of Antimicrobial Disks t h a t s h o w e d n o v i s i b l e g r o w t h o f The modified disk diffusion method was microorganisms were plated out on NA to used to evaluate the antimicrobial activity of investigate microbial growth.After 24h isothiazolone biocide against the test bacterial incubation, the MBC was defined as the lowest isolates.This method was carried out using concentration killing> 99.9% of the isolate.Mueller Hinton agar (MHA) media.Sterile filter paper discs were immersed into the biocides in a RESULTS sterile glass Petri dish in order to embed the Isolation of Marine Fouling Bacteria from biocides onto the perforated paper discs (Shirley Scrapings of Boat Hulls et al., 2010).
Following isolation and 24 h aerobic 0 incubation at 37 C, bacterial growths were Susceptibility Tests observed and counted by the number of visible To carry out the susceptibility test, colonies using a colony counter.The bacterial aliquots (0.1ml) of the standardized inoculum -5 population density ranged from 1.2 x 10 CFU/g was seeded uniformly onto the petri dish MHA The biocides were tested against S. after the incubation period and measured in aureus, P. mirabilis and P. aeruginosa for the millimeter.
antibacterial efficacy which resulted in formation of varying zones of inhibition when compared Determination of Minimum Inhibitor y with the control plates.The diameter of Concentration (MIC) and Minimum Bactericidal inhibition zone of biocide (24.75 mm) against P. Concentration (MBC) mirabilis showed significant potency as In order to determine the bacteriostatic compared to S. aureus (4.64 mm) and (12.75)P. and bactericidal effects of the isothiazolone aeruginosa (Table 1).There was no inhibition biocide on the isolated bacteria, a loopful each of zone observed in the control plates.The overnight cultures of the test isolates on NA was isothiazolone biocide exhibited antibacterial inoculated into sterile peptone water and properties against all the tested isolates in the incubated aerobically at 37°C for 24 h to achieve order: P. mirabilis > P. aeruginosa> S. aureus.an exponential phase.Subsequently, bacterial This study therefore demonstrates that concentration was determined by diluting with isothiazolone biocide can be very effective and peptone water to give a cell concentration of potent against common bacterial isolates 8 approximately 10 colony forming units occurring on boat hulls.The MIC and MBC values (CFU/mL) corresponding to the 0.5 McFarland ranged between 5 and 15 µl/ml and between 15 standard.The MIC of the isothiazolone biocide and 20 µl/ml for P. mirabilis, P. aeruginosa and S. for individual isolates was performed by the aureus respectively (Table 2).modification of the broth dilution method based 1ml) from selected dilutions (10 and 10 ) p y r u v a t e d e h y d r o g e n a s e , s u c c i n a t e were inoculated on nutrient agar (NA) plates.dehydrogenase, NADH dehydrogenase, lactate 10 CFU/g.The mean bacterial and was spread over the surface of the agar with population density observed is displayed in Fig. a sterile hockey stick.With a sterile forceps, the 1. impregnated disks were carefully placed in the middle of the MHA petri dishes.Control plates Antibacterial Effect of Isothiazolone on Cell had no biocide.The plates were incubated o aerobically at 37 C for 24 h.The zones of Viability inhibition surrounding the disks were observed

Fig. 1 .
Fig. 1.Mean Bacterial Population Density of Scrapings from Twenty Boat Samples