*4.5. Statistical Analysis*

Data from FA profiles were square-rooted transformed, and a Bray-Curtis matrix was assembled. A one-way PERMANOVA was used to test for differences between the FA profiles (for both all individual FAs, as well as FA classes) of Ascidiacea and seaweeds originating from +Org and −Org, with "sampling location" being used as a fixed factor. The statistical significance of variance components was tested using 999 permutations of unrestricted permutations of data, with an a priori chose significance level of α = 0.05. Individual differences in the relative abundance of FA (whose values recorded > 5% of the

total pool of FA in at least one of the biological matrices surveyed), FA classes, ∑*n*-3, ∑*n*-6, and the ∑*n*-3/∑*n*-6 ratio from +Org and −Org were compared by either a t-test or the nonparametric Mann-Whitney U rank comparisons if samples were not normally distributed. A MDS was used to graphically visualize overall patterns and relationships between the different biological matrices survey. A SIMPER analysis was used to determine which FAs contributed the most to similarities and dissimilarities within Ascidiacea and seaweeds, at a cut-off of 50%. All analyses were performed using the PRIMER 6 + PERMANOVA© software (software package from Plymouth Marine Laboratory, Plymouth, UK).

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/10 .3390/md19080469/s1, Table S1: 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. BCFA: Branched fatty acids, SFA: saturated fatty acids, MUFA: monounsaturated fatty acids, PUFA: polyunsaturated fatty acids. n.d: not detected; Table S2: Summary of the environmental parameters measured at the time of sampling in locations with versus without the influence of organic-rich waters from fish farming activities (+Org or −Org, respectively). Values are expressed as a percentage and are averages of three replicates (n=6) ± SD; Table S3: Nutritional composition of the formulated fish feed provided to the fish at the fish farming location (+Org).

**Author Contributions:** Funding acquisition, A.I.L. and R.C.; investigation, L.M.; methodology, L.M. and E.d.C.; resources, M.R.D. and M.H.A.; supervision, A.I.L. and R.C.; validation, M.R.D., E.d.C., M.H.A., A.I.L. and R.C.; writing—original draft, L.M.; writing—review and editing, L.M., A.I.L. and R.C.. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by project "AquaMMIn—Development and validation of a modular integrated multitrophic aquaculture system for marine and brackish water species" (MAR-02.01.01-FEAMP-0038) co-funded by Portugal 2020 and the European Union through Mar2020, the Operational Programme (OP) for the European Maritime and Fisheries Fund (EMFF) in Portugal. We also acknowledge FCT (Portuguese Foundation for Science and Technology) for the financial support to L. Marques through a PhD grant (PD/BD/127918/2016). Thanks, are also due to CESAM (UIDP/50017/2020+UIDB/50017/2020), to FCT/MEC through national funds, and the co-funding by the FEDER, within the PT2020 Partnership Agreement and Compete 2020.

**Institutional Review Board Statement:** Not applicable.

**Data Availability Statement:** The data presented in this study are included in the corresponding sections throughout the manuscript.

**Acknowledgments:** The authors are grateful to ALGAplus-Produção e Comércio de algas e seus derivados, Lda. for supplying the ascidians and seaweeds samples for this work.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

