*3.6. Antimicrofouling Evaluation*

### 3.6.1. Bacterial Growth Conditions

Five marine bacterial species were used as models to assess antimicrofouling activity. The biofilm-forming marine bacteria *Marinobacter hydrocarbonoclasticus* DSM 8798 (ATCC 49840), *Cobetia marina* DSM 4741, *Phaeobacter inhibens* DSM 17,395, and *Pseusooceanicola batsensis* DSM 15,984 were obtained from DSMZ (Leibniz Institute DSMZ—German collection of Microorganisms and Cell Cultures) and *Micrococcus luteus* [55]. Cultures were routinely grown in liquid marine broth (Carl Roth GmbH, Karlsruhe, Germany) with agitation (180 rpm) or on agar-supplemented marine broth, at 28 ◦C (*M. hydrocarbonoclasticus* and *C. marina*) or 30 ◦C (*P. inhibens* and *P. batsensis*). *M. luteus* was maintained in Brain Heart Infusion broth (BHI, Becton Dickinson, GmbH, Heidelberg, Germany) with agitation (180 rpm) or on agar-supplemented BHI, at 37 ◦C.

### 3.6.2. Antibacterial Activity Evaluation Assays

The antibacterial activity of the napyradiomycins (**1**–**12**) was assessed in 96-well polystyrene flat bottom microplates (Nunclon Delta Surface, Thermo Scientific, Roskilde, Denmark) following previously reported procedures [42]. For initial screening, bacterial overnight cultures were diluted to an optical density (OD600nm) of 0.2 and incubated statically at 28 ◦C (*M. hydrocarbonoclasticus, C. marina*), 30 ◦C (*P. inhibens, P. batsensis*) or 37 ◦C (*M. luteus*) in a 96-well microplate in the presence or absence of 31.25 μg/mL of the napyradiomycins, solubilized in DMSO. After 24 h (*M. hydrocarbonoclasticus, C. marina*) or 48 h (*P. batsensis, P. inhibens, M. luteus*) incubation, the OD600nm was determined (Molecular Devices, Spectra Max 190). The napyradiomycins which showed antibacterial activity at a concentration of 31.25 μg/mL were then tested at lower concentrations (2-fold serial dilutions: 15.60, 7.81, 3.91, 1.95, and 0.98 μg/mL), and the protocol was repeated as described above. The percentage of growth inhibition was calculated as the amount of growth relative to that of the bacterial species without added compounds (with the same amount of DMSO added). CuSO4 (5 μM), a potent antifouling agen<sup>t</sup> used in antifouling paints, was used as reference.

All assays were performed in triplicate, and results are representative of the average and standard error of the mean (SEM). Statistical analysis was performed in GraphPad Prism 8.0.2 (San Diego, CA, USA), using one-way ANOVA followed by a Dunnett's multiple comparisons test against the control (species grown with the same amount of DMSO added).

### 3.6.3. Antibiofilm Activity Evaluation Assays

The antibiofilm activity of napyradiomycins (**1**–**12**) against the five marine bacterial species was assessed in 96-well polystyrene flat bottom microplates (Nunclon Delta Surface, Thermo Scientific, Roskilde, Denmark) as previously reported [42]. For initial screening, bacterial overnight cultures were diluted to an optical density (OD600nm) value of 0.2 and incubated statically at 28 ◦C (*M. hydrocarbonoclasticus, C. marina*), 30 ◦C (*P. inhibens, P. batsensis*) or 37 ◦C (*M. luteus*) in the 96-well microplate in the presence or absence of 31.25 μg/mL of napyradiomycins, solubilized in DMSO. After 24 h (*M. hydrocarbonoclasticus, C. marina*) or 48 h (*P. batsensis, P. inhibens, M. luteus*) incubation, the OD600nm was determined. The planktonic cells and media were discarded, and the wells were washed twice with deionized water. The biofilm was fixed for 1 h at 60 ◦C and stained with crystal violet 0.06% for 10 min. The dye was discarded, and the wells were again washed twice with deionized water. The stained biofilm was solubilized with 30% acetic acid and the OD600nm was determined. The napyradiomycins which showed antibiofilm activity at a concentration of 31.25 μg/mL were then tested at lower concentrations (2-fold serial dilutions: 15.60, 7.81, 3.91, 1.95, 0.98 μg/mL), and the protocol was repeated as described above. The percentage of biofilm inhibition was calculated as the amount of biofilm relative to that of the bacterial species without added compounds (with the same amount of DMSO added). CuSO4 (5 μM), a potent antifouling agen<sup>t</sup> used in antifouling paints, was used as reference.

All assays were performed in triplicate, and results are representative of the average and standard error of the mean (SEM). Statistical analysis was performed in GraphPad Prism 8.0.2, using one-way ANOVA followed by a Dunnett's multiple comparisons test against the control (species grown with the same amount of DMSO added).

### *3.7. Antimacrofouling Evaluation: Mussel Larvae Mytilus Galloprovincialis Acute Toxicity Assay*

Mussel (*M. galloprovincialis*) adhesive larvae (plantigrades) were used to assess in vivo the antifouling activity of napyradiomycins (**1**–**12**) towards macrofouling. Juvenile mussel aggregates were collected at the intertidal rocky shore during low spring tides, at Memória beach, Matosinhos, Portugal (41◦13-59" N; 8◦43-28" W). At the laboratory, immediately before the bioassays, mussel plantigrade larvae were screened and isolated from the juvenile aggregates using a binocular microscope (Olympus SZX2-ILLT, Hamburg, Germany) and washed with filtered seawater to remove organic debris. Only competent plantigrade larvae (those showing foot exploratory behavior) were selected and used in the exposure bioassays following previously validated procedures [70,71]. Plantigrades were exposed in 24-well polystyrene plates for 15 h in the darkness at 18 ◦C. DMSO was used as solvent for crude extracts, fractions and pure compounds stock and working solutions. Working solutions were prepared by successive dilutions of stock solutions in DMSO and then diluted in filtered seawater to obtain the test solutions. DMSO concentration in test solutions was always 0.1%. Four well replicates were used per condition with five larvae per well. Two negative controls, one with ultra-pure water and the other with DMSO 0.1% were included in all bioassays, as well as a positive control with 5 μM CuSO4 (a potent antifouling agent). Anti-settlement bioactivity was determined by the presence/absence of fixed byssal threads produced by each individual larvae for all the conditions tested. Napyradiomycins were tested at 5 μg/mL and those showing anti-settlement activity were tested at higher and lower successive concentrations (12, 6, 3, 1.5, 0.75, and 0.375 μg/mL) for the determination of the semi-maximum response concentrations (EC50) that had an anti-settlement e ffect in mussel larvae. Chi-square Pearsons goodness-of-fit test was applied to data in order to determine EC50. Significance was considered at *p* < 0.01 for all analyses, and 95% lower and upper confidence limits [95% LCL; UCL] were presented. Therapeutic ratio (LC50/EC50) was used to evaluate the e ffectiveness vs. toxicity of compounds [19,21].

### *3.8. In Silico Environmental Toxicity Assessment*

The in silico toxicity evaluation was done using the Toxicity Estimation Software Tool (T.E.S.T.) [63], https://www.epa.gov/chemical-research/toxicity-estimation-software-tool-test, that was developed to allow users to easily estimate toxicity using a variety of QSAR methodologies. In accordance with the European Union Directive 2001/59/EC and the Regulation on the Classification, Labelling and Packaging of Substances and Mixtures (CLP) 1272/2008, a substance can be classified as "harmful", "toxic", and "very toxic" to aquatic organisms depending on the 96-hour LC50 for fish (e.g., fathead minnow), 48 hour LC50 for daphnids (e.g., *Daphnia magna*), and others assays such as 72-hour IC50 for algae or 40 hour IGC50 for protozoans (e.g., *Tetrahymena pyriformis*). If IC50 or LC50 or IGC50 are below 1 mg/L, a substance is classified as "very toxic to aquatic organisms" (danger symbol N, risk phrase R50). If the values obtained for toxicity are between 1 and 10 mg/L, a substance is classified as "toxic to aquatic organisms" (danger symbol N, risk phrase R51). A substance is classified as "harmful to aquatic organisms" if the end points obtained are between 10 and 100 mg/<sup>L</sup> (risk phrase R52). Classification is also based on the assessment of ready biodegradability or bioaccumulation potential (i.e., bioconcentration factor, BCF). If BCF ≥100, a compound is classified as "may cause long-term adverse e ffects in the aquatic environment" (risk phrase R53) [64,65]. The acute toxicity estimate (ATE) categories, identified by the CLP regulation, depend on the Oral rat LD50 [66]. The four ATE thresholds are; a) category 1, ATE ≤ 5 mg/Kg, a substance is classified as "Fatal if swallowed", b) category 2, 5 < ATE ≤ 50 mg/Kg, a substance is classified as "Fatal if swallowed", c) category 3, 50 < ATE ≤ 300 mg/Kg, a substance is classified as "Toxic if swallowed", d) category 4, 300 < ATE ≤ 2000 mg/Kg, a compound is classified as "Harmful if swallowed" and e) category 5, ATE > 2000 mg/Kg, a substance is

classified as "may be Harmful if swallowed". Mutagenicity, carcinogenicity and reproductive toxicity are some of the most important endpoints to evaluate toxicity towards humans. Mutagenic toxicity can be experimentally assessed by various test systems; the most common is the Ames test, which makes use of a genetically engineered *Salmonella typhimurium* and *Esherichia coli* bacterial strains [67].
