Synthesis of isonitrosoacetanilides and stock-solution preparation
To a 500 mL round-bottom flask were added 0.915 mol of sodium sulfate, 120 mL of distilled water, 0.109 mol of chloral hydrate, 0.079 mol of hydroxylamine sulfate dissolved in 50 mL of distilled water, 0.1 mol of the respective aniline in 60 mL of distilled water and 8.6 mL of concentrated hydrochloric acid. The reaction mixture was gradually heated to 70º C. After adding 100mL of ethyl alcohol, the mixture was heated under reflux until TLC indicated that formation of isonitrosoacetanilide was complete. The mixture was poured onto ice/H2O and the solid isonitrosoacetanilide formed was collected by filtration and washed with water (Garden et al 1997, Silva et al 2011).
Assays were performed using 48-well polystyrene plates (Barloworld Scientific Ltd., Sandton, South Africa). Each compound was first dissolved in a small amount of dimethyl sulfoxide (DMSO) (final concentration = 0.1%) that was first diluted with filtered 0.22 µm and autoclaved seawater 25 PSU (FSW) to achieve stock-solution (2.4 mg mL− 1). The active compounds were diluted with FSW to get 0.625–1.25–2.5–5 – 10 µg mL− 1 (based on Chen et al. 2014). This range was chosen followed Kitano et al. (2011). In all assays 0.1% DMSO served as the solvent control (sC), while FSW as negative control (−C).
The bacterial biofilm consortium were represented by Proteobacteria and Bacterioidetes phyla (98%): Vibrio (24%), Neptuniibacter (16%), Phaeobacter (13%), Alteromonas (11%), Oceanospirillum (10%), Pseudoalteromonas (7%), Methylophaga (7%), Pseudomonas (7%), Oleibacter (5%), and Marinomonas (3%) (Agostini et al., 2019, 2020).
Planktonic and Biofilm Bacteria assays
Crystal violet assay for the estimation of bacterial biomass inhibition and eradication. The ability of the compounds to prevent bacterial adhesion and EPS formation on a virgin surface (inhibition of planktonic and biofilm biomass) and to destroy an established marine bacterial biofilm (eradication of biofilm biomass) were assessed using four replicates per treatment using the crystal violet assay (Agostini et al., 2019b, 2020).
For the biomass inhibition assay, 100 µL of bacterial inoculum, 100 µL of FSW, and/or compounds stock-solution were mixed in 96-well plates (Barloworld
Scientific Ltd., Sandton, South Africa) and incubated at 25°C for 48 h in the dark (OD570 = 0.002). For the biomass eradication assay, 200 µL of bacterial inoculum
(OD570 = 0.002) was incubated at 25°C for 24 h in the dark in 96-well plates. The supernatant in the wells was removed and the compounds were added to these wells and incubated at 25°C for 24 h in the dark.
The planktonic bacterial biomass (free-living bacteria) was estimated by the difference in the absorbance at the beginning (OD600 = 0.002) and end of the incubation. For biofilm assays, the content of the wells was removed, and washed three times with sterile saline solution. The attached bacteria were heat-fixed at 60°C for 1 h. The biofilm layer formed was stained with 0.4% w/v crystal violet for 15 min at 20°C and the plates were washed four times with tap water. The stain bound to the cells was solubilized with 99.5% ethanol (Sigma-Aldrich Co., St. Louis, MO, USA) for 30 min, and the absorbance was measured (Malafaia et al., 2017) using the Spectramax M2e Multimode Microplate Reader (Molecular Devices San Jose, CA, USA).
Adult barnacles from Istituto per lo studio degli impatti Antropici e Sostenibilità in ambiente marino (IAS) - CNR were maintained in aerated, filtered (0.45 µm) natural seawater at 20°C, on a 16-h:8-h light-dark cycle. They were fed every two days with Artemia salina (50 to 100 mL; 200 larvae mL-1) and Tetraselmis suecica (100 to 200 mL; 2 × 106 cells mL-1). The seawater was changed three times per week, and the barnacles were periodically rinsed with fresh water to remove epibionts or debris.
To obtain the nauplii for cyprid cultures, the adults were left to dry for 30 min to 40 min and then immersed in fresh seawater. The hatched nauplii were attracted to a light source and collected using a Pasteur pipette. They were reared to the cyprid stage as described in Faimali et al. (2003), by keeping them at 28°C in natural filtered (0.22 µm) seawater and feeding them three times a week with Tetraselmis suecica (2 × 106 cells mL− 1). In these conditions nauplii reach the cyprids stage in 5–6 days. The cyprids were harvested by filtration and aged for 4 days prior to use, in filtered (0.45 µm) natural seawater at 4°C in the dark (Rittschof et al. 1992).
Amphibalanus amphitrite cyprid settlement and mortality assay
To evaluate the macrofouling settlement inhibition was used barnacle cyprids of A. amphitrite. In the assays, the treatments were conducted in 24-well microplates (Barloworld Scientific Ltd., Sandton, South Africa) by adding 20 to 25 cyprids per well, with each well containing 2 mL of medium, representing the treatments. The test plates were sealed to prevent evaporation and incubated at 28°C in the dark. The settlement and the mortality were evaluated after 72 h of incubation. The larvae were examined under a dissecting microscope, to record the number of dead and permanently attached individuals. The experiments were terminated by the addition of three droplets of 40% formaldehyde into each test well and the counting of the settled and non-settled larvae. The results were expressed as the percentages (± standard error) of the settlement of the total number of larvae incubated (Piazza et al. 2014).
Amphibalanus amphitrite nauplii II swimming alteration and mortality assay
Acute (mortality) and sub-acute (immobility) toxicities were assessed using II stage nauplii A. amphitrite. All of these tests were performed in 48-well microplates (Barloworld Scientific Ltd., Sandton, South Africa) with 10–15 nauplii II per well, each well containing 1 mL of medium. The organisms were evaluated after 24 h and 48 h of incubation at 20°C in the dark. After the exposure the larvae were examined under a dissecting microscope, and the number of dead and not swimming larvae was recorded (Piazza et al. 2014). Larvae that were completely motionless (unable to change their barycenter position or move any appendages in 10 s) were counted as dead, while the larvae that could not swim (unable to change their barycenter position but move appendages) were counted as having a swimming alteration (Agostini et al. 2019b, 2020). The results were expressed as the percentages (± standard error) of the settlement of the total number of larvae incubated (Piazza et al. 2014).
For the bacterial biofilm data, the plant extract that killed planktonic bacteria and consequently prevented bacterial adhesion and biofilm formation was called antibiotic, while the plant extract that prevented bacterial adhesion without killing planktonic bacteria was called antibiofilm (Agostini et al., 2019, 2020). Variance analysis (one-way ANOVA) was used to evaluate possible differences in the bacterial density and biomass (response variables) according to different treatments (predictive variables) after confirming adherence to all the statistical assumptions required by ANOVA (continuous data; normal distribution of residuals, assumption of homogeneity of variances/covariances and mean and variances independence) (Gotelli & Ellison, 2013). Generalized Linear Models (GLM) were used to evaluate settlement and toxicological effects (response variables) of the treatments (predictive variables). Binomial distribution was applied to the logit link function, where Y = n = number of mortalities/swimming alteration/settlement of the total of ‘‘n individuals’’ in the experimental units (Lopes et al., 2018). For toxicity, the tests were considered acceptable when the mortality in the control was < 10% (Garaventa et al., 2010). Tukey’s post hoc test was used when the alternative hypotheses from ANOVA and GLM were acceptable through the multcomp package (Hothorn et al., 2008). Differences were considered significant when the calculated p value was less than 0.05. Analyses were performed on R (version 3.5.1) (R Core Team, 2019). For toxicological assays, the median endpoint values (LC50 and EC50) and related 95% Confidence Intervals were calculated using Spearman and Karber analysis (Finney 1978). The median values were expressed as LC50 for mortality (the concentration able to cause the mortality of the 50% of the tested population) and EC50 for mobility alteration (the concentration able to cause an alteration of larvae mobility for 50% of tested population). We consider immobility (EC) the sum between mortality and swimming alteration.