**2. Results**

### *2.1. Anti-Fouling E*ff*ects of the Crude Extracts*

Antifouling activity of the sea cucumber crude extracts was assessed by measuring biomass and attachment of the diatom *C. closterium*. To assess suspended algal biomass, chlorophyll a *(Chl a)* was extracted from the water samples, while *Chl a* content of diatoms attached to the substrate was used to evaluate diatom attachment. *Chl a* measurements are well established as a proxies for monitoring water quality, assessing phytoplankton biomass, and estimating primary production [61–63], while fluorometric measurements of *Chl a* concentrations are an efficient proxy to monitor the total biomass of diatoms in the water column and on the substrate. To determine the anti-fouling effects of the holothurian's crude extracts, a logarithmic response ratio (LRR; see Section 4.1.5) of measured *Chl a* concentrations was calculated. Negative LRR reveals an anti-fouling effect of the extract with less *Chl a* in the treated compared to the control samples, while a positive LRR indicates a higher *Chl a* concentration and thus an increase in algal growth in the treatments compared to the control samples.

Measurements of *Chl a* concentration of the suspended cells in the water and the attached cells at the flasks surface showed that the sea cucumbers crude extracts had a concentration-dependent effect on growth and settlement of *C. closterium* (Figure S1A–F). The LRR supports this finding (Figure 3A,B), showing the highest negative effect (*p* < 0.05) on diatom growth in the water column at the highest extract concentrations (150 μg mL−1, Figure 3A), except for extracts from *H. whitmaei* and *H. hilla* where no negative effects could be observed (*p* = *0.371* and *p* = *0.65*, respectively; Table S1). *Actinopyga spp.* and *Bohadschia* extracts (except *B. vitiensis*) exhibited negative LRR at 15 μg mL−<sup>1</sup> concentration, indicating significant anti-fouling effects. Extracts of the genera *Holothuria* (except *H. atra*) had no inhibitory effects at the same concentration. At the lowest concentration (1.5 μg mL−1; Figure 3A), all the crude extracts showed a positive LRR, except *B. argus* and *A. echinites* extracts, which had significant inhibitory activity toward the tested diatom in the water column.

Similar to the LRR in the water column, the highest crude extract concentrations (150 μg mL−1) inhibited diatom settlement (Figure 3B). The treatment containing 15 μg mL−<sup>1</sup> of extract of the genus *Holothuria* stimulated diatom settlement, whereas *Bohadschia* (except *B. vitiensis*) and *Actinopyga* extracts suppressed it. At the lowest concentration (1.5 μg mL−1) all crude extracts (except *B. vittiensis*) showed a significant inhibition on diatom settlement (Table S1).

**Figure 3.** Logarithmic response ratio (LRR) of *C. closterium* after exposure to three different concentrations (150, 15 and 1.5 μg mL−1) of nine sea cucumber extracts in total (genera *Holothuria*, *Bohadschia* and *Actinopyga*) for (**A**) suspended cells in the water and (**B**) attached to the surface of the incubation flask. Significant differences compared to the control (CT = control) are shown in Table S1.

### *2.2. Saponin Profile of the Crude Extracts*
