*2.3. Fish Feed*

A total of 26 FAs were identified in fish feed (Supplementary Information Table S1) (Table 1). MUFA was the most abundant FA class for fish feed (44.8%) (Figure 1) with a major contribution of FA 18:1(*n*-7+*n*-9) (36.0%) (Table 1). SFA and PUFA presented similar values (25.7% and 29.5%, respectively). The *n*-3/*n*-6 ratio obtained was 0.63, indicating higher amounts of *n*-6 FAs. The MDS plot (Figure 2) revealed that the FA profile of fish feed is more similar to the FA profile of Ascidiacea from +Org than from −Org. SIMPER analysis of the FA profiles of fish feed and Ascidiacea (Table 3B) revealed higher dissimilarities with specimens originating from −Org. For Ascidiacea, EPA was the main responsible for such differences.

**Figure 2.** Multidimensional scaling (MDS) ordination plot comparing the fatty acid profiles between specimens of ascidians (Ascidiacea) (A) and seaweeds (sea lettuce, *Ulva* spp. (U) and bladderwrack, *Fucus* sp. (F)) sampled in locations with versus without the influence of organic-rich effluents from fish farming activities (+Org or −Org, respectively) and the formulated fish feed (FF) most commonly supplied in fish farming activities in the study location.

#### **3. Discussion**

To the authors' best knowledge, the present study is the first approach reported in the scientific literature to screen for health-promoting FAs in ascidians grown under the influence of fish farming organic-rich effluents. From the total pool of FA identified in Ascidiacea (42 FAs), only 4 of these biomolecules (16:0, 18:1(*n*-7+*n*-9), EPA, and DHA) represented average values above 10% of the total pool of FA. These findings share similarities with those reported from previous works screening the FA profile of ascidians [37,48–50]. The FAs 18:1(*n*-7+*n*-9) and 18:2*n*-6 also displayed higher values in +Org, near twice the ones recorded for −Org. Considering that these FAs accounted for 53% of the fish aquafeed FA pool, it is likely that ascidians may selectively retain these FAs in their tissues. The higher levels of *n*-3 FAs present in the −Org resulted in a higher *n*-3/*n*-6 ratio, with FAs 18:4*n*-3, EPA, and DHA being the main contributors to this trend. This finding is consistent with Monmai et al. [35], as these authors verified that in the edible ascidian *Halocynthia aurantium n*-3 FAs were present in much higher levels than *n*-6 FAs. Likewise, Zhao and Li [37] documented that tunics and inner body tissues of ascidians *Halocynthia roretzi*, *Styela plicata*, *Ascidia* sp. and *Ciona intestinalis* presented higher levels of *n*-3 FAs.

*Ulva* spp. and *Fucus* sp. presented some similarities in their FA profiles, with 16:0 and 18:1(*n*-7+*n*-9) displaying the highest relative abundances in the total pool of FAs recorded in both locations. This finding is in line with previous studies [51–53]. Our results on the profiling of unsaturated FAs (MUFA+PUFA) are fully aligned with those reported by Herbreteau et al. [54], who reported the FA composition of five species of seaweeds and verified that unsaturated FAs accounted for more than 50% of the total pool of FAs, with this proportion reaching up to 75% for *Fucus* sp. Silva et al. [55] focused on ten brown seaweeds also verifying important amounts of unsaturated FAs. In addition, our study recorded 46% to 49% of SFA in *Ulva* spp., unlike Lopes et al. [4] who have reported about half of these values for the same seaweed species (≈24%). Yet, the values of FA classes reported for *Fucus* sp. by Lopes et al. [4] are very much in line with the ones reported in the present work. Several studies [4,55,56] have mentioned that despite lipid content representing a minor fraction of seaweeds, it features levels of *n*-3 PUFAs worth being investigated. Our results validated the presence of EPA in *Fucus* sp., but not DHA, and no traces of either of these FAs were detected in *Ulva* spp. These latter values correlate

fairly well with Pereira et al., [57] with *Ulva* spp. also presenting higher proportions of FA 18:3*n*-3, and thus, further supporting the idea that seaweeds do display an *n*-3/*n*-6 "healthy" ratio.

Several studies [1,5,6,30,58] have reported an increase in the use of *n*-6 PUFA-rich land-based ingredients and oils in aquafeed formulations sometimes leading to an inverted *n*-3/*n*-6 ratio in fish aquafeeds. Under organic-rich effluents, the biochemical profile of extractive species will most likely be shaped by the prevalence of these ingredients [16]. However, the availability of natural nutrients [59], sampling location, and season [55], amongst other factors, must be taken into consideration when profiling the FAs of marine species, as they too can modulate their biochemical profile and findings being reported results must be interpreted with care. Kim et al. [52] demonstrated how temperature, salinity, light, and nitrogen levels influence the level and profile of lipids present in the brown seaweed *Fucus serratus*. Similar findings were reported by Glencross [23] who emphasized how the hydrological source is a primary factor weighing in on the differences in FA requirements. This trend can extend to a multitude of marine organisms of interest for production under an IMTA framework, such as polychaetes [60,61], isopods [62], bivalves [63,64], and several fish species [65].

In conclusion, the present study demonstrated that Ascidiacea presented high values of EPA (17.8% in +Org; 20.4% in −Org) and DHA (8.8% in +Org; 11.9% in −Org) and can be considered as a potential new bioresource for *n*-3 long-chain FAs. The organicrich effluent originating from fish farming systems can indeed shape the lipid profile of extractive species being employed in IMTA frameworks, whether as a consequence of direct consumption of available organic nutrients in dissolved and particulate form, as in the case of ascidians, or indirectly from de novo FA synthesis as in the case of seaweeds uptaking dissolved inorganic nutrients. The use of extractive species to maximize the use of ingredients present in formulated aquafeeds employed to farm marine finfish and shrimp can be considered as a pathway towards more sustainable and efficient aquaculture practices and have the potential to generate biomass with the potential to deliver important biomolecules for multiple biotechnological applications [66]. Our findings clearly point towards the need to further investigate the biochemical profile, particularly the FA profile of extractive species used in IMTA systems, as an approach to sequester valuable healthpromoting FAs that will otherwise be lost to the aquatic environment through the effluents of fish farms.

#### **4. Materials and Methods**

#### *4.1. Study Areas*

Ria de Aveiro is a shallow coastal lagoon in the west margin of mainland Portugal that inholds the Vouga river estuary and presents a complex and irregular geometry. This coastal lagoon has four main channels emerging from the sea entrance: S. Jacinto-Ovar, Espinheiro, Ílhavo, and Mira channel (Figure 3). The first sampling location surveyed was located at Mira channel (40◦36'51" N, 8◦44'25" W) without the influence of organic-rich effluents from fish farming activities and is herein referred as −Org. The second sampling location surveyed was located at a land-based semi-intensive fish farm (40◦36'43" N, 8◦40'43" W) supplied by Ílhavo channel's waters. An IMTA framework is employed in this location, on which European seabass and Gilthead seabream are produced in earthen ponds and seaweeds are produced in tanks supplied with organic-rich waters from these earthen ponds. This location will be referred to as +Org. Both channels of this coastal lagoon present strong salinity gradients with very low values at their upper reaches. Salinity, temperature, dissolved oxygen, and pH were registered in situ at the time of sampling. Environmental parameters are summarized as Supplementary Information (Table S2).

**Figure 3.** Sampling locations at Ria de Aveiro coastal lagoon (Portugal): (**A**) located in Mira channel (40◦36'51" N, 8◦44'25" W) and without the influence of organic-rich effluents from fish farming activities (−Org); and (**B**) located at a land-based semi-intensive fish farm (40◦36'43" N, 8◦40'43" W) supplied by Ílhavo channel's waters employing an IMTA framework where seaweeds are produced in tanks supplied with organic-rich waters from earthen ponds stocked with fish (+Org).
