**1. Introduction**

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Marine organisms are commonly perceived as a rich source of *n*-3 fatty acids (FA) [1–4] whose consumption ensures health-promoting benefits against cardiovascular and neurological diseases. Additionally, consumers also acknowledge the anti-inflammatory, anticoagulation, anti-oxidative properties (among others) of *n*-3 FA originating from seafood, making them paramount for human nutrition [5–8]. As a result of the fast-growing trend of the world population [8,9] and the high request for nutritious and healthy marine food [1,10,11], aquaculture activities are facing a major challenge in recent years to keep up with an ever-growing demand. Proportionally, there is also a growing focus on the improvement of aquaculture efficiency, as well as the promotion of environmentally and financially sustainable practices [12–15]. As an example of this ongoing effort, one can refer to the reduction of the levels of marine-based ingredients, such as fishmeal and fish oil, in the formulation of aquafeeds for marine species aquaculture (namely finfish and shrimp) [11,16]. Indeed, a growing proportion of marine-based ingredients have been partially replaced by land-based ingredients (e.g., wheat, soy, corn) [17–19] and oils (e.g., palm

**Citation:** Marques, L.; Domingues, M.R.; da Costa, E.; Abreu, M.H.; Lillebø, A.I.; Calado, R. Screening for Health-Promoting Fatty Acids in Ascidians and Seaweeds Grown under the Influence of Fish Farming Activities. *Mar. Drugs* **2021**, *19*, 469. https://doi.org/10.3390/ md19080469

Academic Editor: Bill J. Baker

Received: 9 August 2021 Accepted: 20 August 2021 Published: 22 August 2021

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oil, soybean oil, sunflower oil) [20,21]. Nonetheless, aquafeeds for marine species production still include marine-based ingredients to achieve desirable FA profiles [22]. These marine-based ingredients, particularly fish oil, are a source of essential FAs, such as *n*-3 long-chain polyunsaturated FAs (PUFA) 20:5*n*-3 eicosapentaenoic acid (EPA), and 22:6*n*-3 docosahexaenoic acid (DHA), which are paramount to ensure the healthy development of species being farmed, and as such, safeguard that these remain a valid source of these important nutrients in human diets [23,24]. Consequently, the aquaculture industry has evolved to develop productive frameworks that target the co-production of extractive species that impair the loss of valuable nutrients (such as *n*-3 long-chain PUFA); this approach has been termed integrated multi-trophic aquaculture (IMTA) and has gained a growing interest in the scientific community [25–28]. These productive systems benefit from the simultaneous farming of species occupying different trophic levels to sequester, recycle and remove excess nutrients originating from uneaten and undigested feed, as well as excretion products [29] present in aquaculture effluents that shape the biochemical content of co-farmed species [10]. Extractive species produced under organic-rich effluents (Org) are responsive to their surrounding environment and experience more or less pronounced shifts in their biochemical composition [2,30,31]. Consequently, FA analysis has become an excellent tool to trace the biochemical fingerprint of aquaculture effluents in aquatic environments and their species [32,33].

Ascidians are marine filter-feeders commonly investigated for marine natural products development, such as anti-cancer and anti-malarial drugs [34]. Knowledge on ascidians' FA profiling is still poorly explored. However, some studies have already confirmed that ascidians present a high *n*-3/*n*-6 ratio [3,35] and high values of EPA and DHA [36], establishing ascidians as a potential new bioresource for *n*-3 fatty acids-rich marine lipids [3, 37,38]. Hassanzadeh [38] concluded that the FA profile of ascidians presented similar values to that of fish oil and, therefore, considered ascidians as a good alternative for fish oil in the formulation of aquafeeds. Additionally, ascidian's biomass may even successfully replace fishmeal in the formulation of aquafeeds [39,40].

The use of seaweeds has been thoroughly explored in IMTA systems [26,41,42]. Seaweed production under this productive framework is receiving growing attention for mass production given their nutritional value and profile in natural bioactive metabolites (particularly with antioxidant properties) [41,43]. Similar to ascidians, seaweeds are considered an important source of *n*-3 long-chain PUFA, especially α-linolenic acid (ALA; 18:3*n*-3) and EPA [4,44], with their potential as ingredients for aquafeed formulations also being increasingly acknowledged [45]. Although the lipid content in seaweed is relatively low (1.27% to 9.13%) [46], these organisms feature high *n*-3/*n*-6 ratios, making them an appealing source of a valuable source of essential FA in health-promoting diets [47].

The present study aimed to compare the FA profile of ascidians (Ascidiacea) and seaweeds (sea lettuce, *Ulva* spp. and bladderwrack, *Fucus* sp.) sampled in a coastal lagoon with versus without the influence of organic-rich effluents from fish farming activities. Additionally, the FA profile of ascidians is also contrasted with that of the most commercially used fish aquafeed employed in the studied location to investigate whether these filter-feeding marine organisms somehow mimicked the FA profile of those aquafeeds when grown under the influence of organic-rich effluents originating from fish farms.
