# Anti-Fouling Properties of Phosphonium Ionic Liquid Coatings in the Marine Environment
## 3 Result and Discussion
### Field Testing Results
#### 3.2.4 Correlation of Anti-Microbial Activity and Marine Anti-Fouling Performance  
Antimicrobial testing was performed to determine whether antimicrobial resistance was linked to the prevention of marine anti-fouling performance. The widely accepted mechanism for marine fouling shown in Figure 1 considers bacterial adhesion the second stage of biofuling [1]. Therefore, we would expect that superior anti-fouling performance would be achieved with antibacterial materials. To study the inhibition capacity of the ionic liquids, trioctylphosphonium samples were exposed to both _S. epidermidis_ (Gram-positive) and _E. coli_ (Gram-negative), and observations of fluorescence and disc diffusion assays were recorded (Supplementary Section S5). Figures 13 and 14 shows the fluorescence readings of the trioctylphosphonium samples against _E. coli_ and _S. epidermidis_ after 24 h of incubation. All the samples showed inhibition against _S. epidermidis_ (Gram-positive), with P\({}_{888}\)[65,28,0] and P\({}_{888}\)[48,48,0] showing the largest inhibition. In contrast, the phosphonium ion gels showed zero inhibition towards _E. coli_. As with the marine anti-fouling, the P\({}_{888}\) series of ion gels showed the most inhibition of _S. epidermidis_ (<50% the growth of the control sample). Interestingly, both ion gel formulations assisted the growth of the Gram-negative _E. coli_, which may have been due to providing phosphorous as a nutrient. These results suggest that antibacterial behaviour (towards Gram-positive bacteria) may contribute to anti-fouling performance. However, the results from disk diffusions assays were less clear. With the exception of sample P\({}_{888}\)[48,48,0], all formulations of the ion gels were ineffective against _E. coli_ and _S. epidermidis_ (Supplementary Materials Figures S33-S36). Disk diffusion assays were performed on all the coated samples using S. _epidermidis_ and _E. coli_. The presence of a halo around the sample P\({}_{888}\)[48,48,0], which was dipped in a Gram-positive bacteria agar plate, concludes that the formulations are effective against Gram-positive bacteria. No halo was present around the samples placed in the Gram-negative bacteria plate. The findings from this work somewhat reflect the 2020 study conducted by [PERSON] et al., who examined the antimicrobial behaviour of more aggressively hydrophobic trialkylphosphonium docusate ion gels [9]. The more hydrophobic ion gels were shown to be effective against both Gram-positive and Gram-negative bacteria (_S. aureus_ and _P. aeruginosa_, respectively). The antibacterial behaviour of both phosphonium and imidazolium ionic liquids appears closely tied to the chain lengths of the alkyl substituents [23,44,45,46]. Figure 12: Graph showing relationship between water contact angle and total fouling rate. The samples are plotted in the decreasing order of free ionic liquid in the formulation. The color gradient (higher free ionic liquid content) is used to differentiate different formulations in trialkylphosphonium ionic liquid gels. Figure 14: The graph shows the relative absorbance readings at 600 nm of trioctylphosphonium doocusate samples with respect to the control sample (where no bacterial growth was inhibited). The sample labeled P888[a,b,c], where ’a’ represents the percentage of the polymerizing group, ‘b’ represents the percentage of free ionic liquid, and ’c’ represents the percentage of copper oxide used in the formulation, was subjected to treatments with both Gram-negative and Gram-positive bacteria. The error bars represent the relative standard deviation of the samples with respect to the control sample. The samples inhibited the growth of S. epidermidis, as the relative absorbance values were smaller than that of the control sample used, whereas there was zero inhibition of _E. coli_ by the samples.