## 2 Results and Discussion
### Comparison of the Action of a Tertiary Amine versus a Quaternary Ammonium Group  
In previous work [16,18,20,22], in order to prevent biofouling, the strategy of immobilizing a biocide on the coating surface was adopted. The quaternary ammonium group is widely known to have biocidal activity [23] therefore it was considered for covalently functionalizing the natural rubber derived oligomers. Several structures were designed and used in the synthesis of polyurethane films or in the synthesis of coatings by radical or cationic photopolymerization. Different biological contact tests showed that the QA group retained its biocide ability even when incorporated in the polymer matrix. This effect was confirmed and discussed in Section 2.1 in the present work, using a different methodology to perform the attachment studies, more quantitative than in the former work. However,  
Figure 3: Effect of hydrogenation or epoxidation of acrylate **(1)**. 33%–67% hydrogenation = oligomer **(3)**; 30%–55% epoxidation = oligomer **(2)**. _Pseudomonas aeruginosa_ **(left)**, _Staphylococcus aureus_ **(middle)**, _Staphylococcus epidermidis_ **(right)**. **(a)**, **(c)**, **(e)** = bacteria suspended in the supernatant media; **(b)**, **(d)**, **(f)** = bacteria attached to the control (polystyrene) or polymer surface. *, difference in values significant at \(p\leq 0.05\), **difference in values significant at \(p\leq 0.01\).  
as the synthesis of the ammonium oligomers requires a certain number of steps, the question arose if the tertiary amino group precursor of the quaternary ammonium would have a similar activity, whilst avoiding the last quaternization step. Moreover the legislation [24] is aiming at the interdiction of the use of QA moieties, because of the toxic effect on the environment in case of ageing of the material and of the consequent release. Therefore we have anticipated shifting towards other biocide groups, even if no release of ammonium salts in the medium occurs with our polymers.  
The amine oligomer **(4)** (Figure 1) is the precursor of the ammonium oligomer **(5)**. It was tested in the same conditions and it displayed a different influence depending on the bacterium (Figure 4). For PA the effect was the most relevant as almost no bacterial cells adhered to the polymer surface and few remained in suspension. For SE there was a pronounced effect in comparison to the control polystyrene but it was slightly less effective than the acrylate alone; for SA there is a small effect in comparison to control and acrylate. If the activity of the amine oligomer **(4)** is compared to the QA oligomer **(5)** (in Figure 2) it is clear that the ammonium has a much stronger effect; however it could be envisioned in future work to obtain surfaces from the mixture of different percentages of oligomers, introducing the amine oligomer **(4)** together with another one containing the biocidal group. An optimal formulation could be found, taking into account the benefit of reducing the number of synthetic steps in view of scale-up and real applications.  
Figure 4: Comparison of basic activity of surfaces from acrylate **(1)** oligomer, from amine **(4)** oligomer, from acrylate mixed with zosteric acid powder (ZA) or with zosteric sodium salt powder (ZAS). _Pseudomonas aeruginosa_ (**left**), _Staphylococcus aureus_ (**middle**), _Staphylococcus epidermidis_ (**right**). (**a**), (**c**), (**e**) = bacteria suspended in the supernatant media; (**b**), (**d**), (**f**) = bacteria attached to the control (polystyrene) or polymer surface. *, difference in values significant at \(p\leq 0.05\), ** difference in values significant at \(p\leq 0.01\).