*2.2. Comet Assay*

The light/dark experimental treatment (here identified as the two-factor variable "treatment") was the only explanatory variable found to significantly modulate DNA damage (Table 2). In accordance, significant differences were found between the L (light) and D (dark) experimental conditions in gills exposed to extracts from the proboscis, albeit only for the 50% concentration (D2), attaining a maximum of ≈56% DNA in tail (Figure 6). In the case, gills exposed to the extract, in the light, revealed highest damage by ca. two-fold on average, relative to the dark condition (Figure 7). Even though pigment extracts from the skin failed to elicit significant differences between experimental conditions, regardless of dilution, the highest level of DNA damage scored in gills exposed to this extract was recorded in gills subjected to light and exposed to the highest (100%) concentration of pigments (termed D1), attaining an average of 51% DNA in tail.

**Table 1.** Summary of the main pigments in PBS extracts from the skin and proboscis of *Eulalia viridis*, based on data retrieved from HPLC-DAD.


[•] and [–] indicate presence or absence, respectively.

**Figure 6.** Comet assay results (as % DNA in tail) from the gills of *Mytilus* exposed to two dilutions of pigment extracts corresponding to the nominal concentrations of 100% (D1) and 50% (D2), plus controls, i.e., gills treated with PBS only, which was the vehicle for pigment extracts (C). Mussels were exposed to the extracts from skin and proboscis mixtures under light (L) and dark (D) conditions. The results are expressed as means + SD. [\*] indicates significant differences between L and D conditions (*t*-test, *p* < 0.05).


1 significant with *p* < 0.05.

**Table 2.** Results from ANOVA for GLM (based on sequential analysis of deviance) for the Comet assay on mussel gills exposed to pigments.

**Figure 7.** Exemplificative Comet fields from mussel gills treated with pigment extracts from *Eulalia* illustrating the differences between light and dark treatments. (**A**) Gills exposed to 50%-diluted extracts from the proboscis (light treatment); (**B**) same as previous but gills subjected to dark conditions, evidencing lower DNA damage, as seen by higher and lower head and tail intensities, respectively, comparatively to the previous.

### *2.3. Daphnia Immobilization Assay*

All explanatory variables ("treatment", "exposure time", "pigment", and "dilution") were found to significantly modulate the immobilization of *Daphnia* (Table 3). Extracts from the skin were responsible for the overall highest immobilization rates, which tended to increase with concentration and total exposure time after the initial 1-h light or dark treatment (Figure 8). However, contrarily to the previous findings, exposure to pigment extracts in the dark tended to cause highest effects either at 24 (Figure 8A) or 48 h (Figure 8B), albeit the effects being more significant for assays conducted with the most concentrated extracts from the skin. Accordingly, the highest immobilization rates resulted from *Daphnia* exposed to the most concentrated (D1) skin pigment extract, followed by exposure to D2, both after 48 h, hitherto the dark treatment attaining a ca. two-fold increase relatively to the light condition. Still, after 24 h of the experiment this effect was almost three-fold higher.

**Table 3.** Results from ANOVA for GLM (based on sequential analysis of deviance) for the *Daphnia* immobilization assay.


1 significant with *p* < 0.01.

**Figure 8.** *Daphnia* immobilization assay after exposure to three dilutions of pigment extracts corresponding to the nominal concentrations of 100% (D1), 50% (D2), and 10% (D3), plus controls (C). *Daphnia* were exposed for 1 h to the extracts from skin and proboscis mixtures under light (L) and dark (D) conditions and analyzed after (**A**) 24 h; (**B**) 48 h. The results are expressed as mean immobilized individuals (out of 20) + SD. [\*] indicates significant differences between L and D treatment conditions (*t*-test, *p* < 0.05).
