**2. Results**

#### *2.1. Ascidiacea*

The FA profile of Ascidiacea revealed a total of 42 different FA (Supplementary Information Table S1). Nonetheless, 4 FAs alone represented more than 50% of the total pool of FAs (Table 1).

**Table 1.** Fatty acid profile of ascidians (Ascidiacea) and seaweeds (sea lettuce, *Ulva* spp. and bladderwrack, *Fucus* sp.) sampled in locations with versus without the influence of organic-rich effluents from fish farming activities (+Org or −Org, respectively), as well as the formulated fish feed (FF) most commonly supplied in fish farming activities in the study location. Values are expressed as a percentage of the total pool of fatty acids and are averages of five replicates (*n* = 5) ± SD. Only fatty acids accounting for at least 5% of the total pool of fatty acids in one of the biological matrices surveyed are presented. SFA: saturated fatty acids, MUFA: monounsaturated fatty acids, PUFA: polyunsaturated fatty acids.


nd: not detected; \* *p* < 0.05; \*\* *p* < 0.001. ∑SFA: 14:0, 15:0, 16:0, 17:0; 18:0, 20:0, 21:0, 22:0, 24:0; ∑MUFA: 15:1, 16:1, 16:1*n*-7, 16:1n-9, 17:1, 17:1*n*-9, 18:1*n*-7+*n*-9, 20:1, 20:1*n*-9, 20:1*n*-7, 22:1*n*-11, 22:1*n*-9, 24:1*n*-9; ∑PUFA: 16:2, 16:2*n*-6, 16:3*n*-3, 16:4*n*-3, 18:2, 18:2*n*-6, 18:3*n*-6, 18:3*n*-3, 18:4*n*-3, 20:2, 20:2*n*-6, 20:3*n*-6, 20:3*n*-3, 20:4*n*-6, 20:4*n*-3, 20:5*n*-3, 22:4, 22:4, 22:5*n*-6, 22:5*n*-3, 22:6*n*-3.

> PERMANOVA test revealed the existence of significant differences in the FA profiles (*p* = 0.006) and FA classes (*p* = 0.011) of Ascidiacea from the two locations surveyed (Table 2). Furthermore, statistical differences were also recorded between all FA classes (Table 1). Concerning the *n*-3/*n*-6 ratio, significant differences were detected between both sampling locations (*p* = 0.002) (Table 1), with higher values being recorded for Ascidiacea sampled at −Org (5.77) (Figure 1). In general, all FAs presented a higher relative abundance at –Org, with the exception of FA octadecenoic acid 18:1(*n*-7+*n*-9), 18:2, 18:2*n*-6, and 20:1*n*-9, which displayed higher abundances at +Org. The FAs EPA and DHA were the two most well-represented FAs (17.8% for +Org and 20.4% for −Org; 8.8% for +Org and 11.9% for −Org, respectively) (Table 1). Furthermore, the relative abundance of FAs 18:1(*n*-7+*n*-9), 18:2*n*-6, and DHA differed significantly between the two locations (Table 1).

> **Table 2.** The results of the permutational multivariate analysis of variance (PERMANOVA) of fatty acids and fatty acid classes of ascidians (Ascidiacea) and seaweeds (sea lettuce, *Ulva* spp. and bladderwrack, *Fucus* sp.) sampled in locations with versus without the influence of organic-rich effluents from fish farming activities (+Org or −Org, respectively). Significant differences were considered at *p* < 0.05 (represented in bold); P(perm): *p*-values based on more than 999 permutations.


**Figure 1.** Fatty acid classes expressed as a percentage of the total pool of fatty acids (only values above 1% were considered) of ascidians (Ascidiacea) and seaweeds (sea lettuce, *Ulva* spp. and bladderwrack, *Fucus* sp.) sampled in locations with versus without the influence of organic-rich effluents from fish farming activities (+Org or −Org, respectively), as well as the formulated fish feed (FF) most commonly supplied in fish farming. BCFA: branched fatty acids, SFA: saturated fatty acids, MUFA: monounsaturated fatty acids, PUFA: polyunsaturated fatty acids.

Branched FAs (BCFA) represented the least abundant FA class identified in specimens sampled from both locations (4.6% for +Org; 5.5% for −Org) (Figure 1). Saturated FAs (SFA) and PUFA demonstrated higher values in specimens from −Org (22.3% and 48.5%, respectively). In addition, monounsaturated FAs (MUFA) values were higher at +Org (33% for +Org and 20.7% for −Org) (Figure 1). Similarity Percentage Species Contributions (SIMPER) analysis (Table 3A) showed that the FA profiles of Ascidiacea originating from the two locations displayed an average dissimilarity of 10.6%, with more than 50% cumulative dissimilarities being explained by the following FAs: eicosenoic acid 20:1*n*-9, 18:1(*n*-7+*n*-9), linoleic acid—LA 18:2*n*-6, and stearidonic acid—SDA 18:4*n*-3.

## *2.2. Seaweeds*

A total of 17 and 24 different FAs were identified for *Ulva* spp. and *Fucus* sp., respectively (Supplementary Information Table S1) (Table 1). The FAs palmitic acid 16:0 and 18:1(*n*-7+*n*-9) were dominant in both seaweeds (37.7% for +Org and 38.1% for −Org; 15.2% for +Org and 15.2% for −Org, respectively). However, some contrasts worth highlighting were also recorded, such as the relative abundance of arachidonic acid (AA) 20:4*n*-6 and EPA in *Fucus* sp. (14.1% for +Org and 15.0% for −Org; 7.7% for +Org and 10.0% for −Org; respectively) that were either not detected or present at trace levels (respectively) in *Ulva* spp. Statistically significant differences were detected in 18:3*n*-3 for *Ulva* spp. (*p* = 0.025), while for *Fucus* sp. FAs 18:1(*n*-7+*n*-9), 18:2*n*-6, 18:3*n*-3, and EPA all differed significantly (*p* = 0.003, *p* = 0.013, *p* < 0.001, *p* < 0.001, respectively). PERMANOVA test showed statistical differences in the mean FA profiles of seaweeds originating from the two sampling locations (*p* = 0.021 for *Ulva* spp.; *p* = 0.013 for *Fucus* sp.), yet only significant differences were seen in the FA classes of *Fucus* sp. (*p* = 0.013) (Table 2), with significant differences being recorded between MUFA and PUFA (*p* = 0.005, *p* < 0.001, respectively) of specimens of this brown seaweed originating from the two sampling locations (Table 1). The *n*-3/*n*-6 ratio also exhibited significant differences between both sampling locations (*p* < 0.001 for *Ulva* spp., *p* < 0.001 for *Fucus* sp.) (Table 1), with higher values being recorded for seaweeds at −Org. The prevailing FA class in *Ulva* spp. was SFA (46.3% for +Org and 48.8% for −Org) (Figure 1), while PUFA registered higher values for *Fucus* sp. (41.6% for +Org; 49.4% for −Org). The non-metric multidimensional scaling (MDS) plot (Figure 2) revealed a

distinct separation between the two seaweeds and the two sampling sites, with similarity values of 59% grouping both FA profiles.

**Table 3.** Summary of SIMPER analysis listing the fatty acids that most contributed to discriminate: (**A**) ascidians (Ascidiacea) and seaweeds (sea lettuce, *Ulva* spp. and bladderwrack, *Fucus* sp.) sampled in locations with versus without the influence of organic-rich effluents from fish farming activities (+Org or −Org, respectively); and (**B**) ascidians from +Org or −Org with the formulated fish feed (FF) most commonly supplied in fish farming activities in the study location. Cut-off percentage: 50%.


SIMPER analysis (Table 3A) revealed that the FA profiles of *Ulva* spp. and *Fucus* sp. display comparable values of dissimilarities between +Org and −Org (5.29% and 5.48%, respectively), with FA 18:0 contributing the most for such differences.
