Global aquaculture production has expanded at an average annual rate of approximately 5.3% over the past three decades, reaching over 90 million metric tons in 2022 and playing an increasingly pivotal role in meeting worldwide demand for animal protein (FAO 2022). Within this trajectory, salmonid aquaculture represents one of the most economically significant and nutritionally valued sectors of the industry (Turchini et al. 2009). Sustaining this growth, however, is contingent upon securing reliable and cost-effective sources of dietary ingredients, most critically lipids, which supply both metabolic energy and physiologically essential fatty acids to farmed fish (Tocher 2003; Turchini et al. 2011).

Historically, fish oil derived from forage fish species has served as the primary lipid source in commercial salmonid feeds, owing to its high content of n-3 long-chain polyunsaturated fatty acids (LC-PUFAs)—principally eicosapentaenoic acid (EPA; 20:5n-3) and docosahexaenoic acid (DHA; 22:6n-3)—and its high energy density (Tocher 2003; Rinchard et al. 2007). These fatty acids are recognized as conditionally essential for salmonids, supporting membrane fluidity, immune competence, osmoregulation, neural development, and reproduction (Sargent et al. 2002; Tocher 2010). However, global fish oil production has remained relatively static over the past two decades, constrained by the finite productivity of marine forage fish stocks and increasing regulatory pressure (FAO 2022). Simultaneously, demand for fish oil in aquafeeds has intensified with the rapid global expansion of salmonid aquaculture, creating a structural supply-demand imbalance that has driven sustained upward pressure on fish oil prices and intensified the search for sustainable alternatives (Tacon and Metian 2008; Tacon et al. 2011).

Plant-derived oils—including soybean, canola (rapeseed), linseed, sunflower, palm, and safflower oils—have been the subject of intensive research as partial or complete fish oil replacements in aquafeeds over the past two decades (Bell et al. 2001; Tocher 2003). These oils offer several practical advantages; they are produced in large volumes from terrestrial agriculture, are available at considerably lower cost than fish oil, have stable and predictable supply chains, and are free from contaminants such as persistent organic pollutants (Gunstone 2011; Shepherd and Jackson 2013). The principal nutritional limitation of plant oils as fish oil substitutes is their lack of EPA and DHA and their enrichment in n-6 PUFAs, particularly linoleic acid (18:2n-6), which can competitively inhibit endogenous LC-PUFA biosynthesis (Tocher 2003; Tocher et al. 2006). Consequently, complete plant oil substitution typically results in a marked reduction of EPA and DHA in fish tissue, with potential implications for the nutritional value of the product for human consumers (EFSA 2012; Sprague et al. 2016).

Numerous studies have demonstrated that partial or complete replacement of fish oil with canola or safflower oil is compatible with normal growth performance in a range of finfish species, including rainbow trout, Oncorhynchus mykiss (Walbaum, 1792) (see Acar and Turker 2018; Yıldız et al. 2018), Atlantic salmon, Salmo salar Linnaeus, 1758 (see Davis and Devine 2023), gilthead sea bream, Sparus aurata Linnaeus, 1758 (see Sánchez-Moya et al. 2020), European sea bass, Dicentrarchus labrax (Linnaeus, 1758) (see Verge-Mèrida et al. 2022), and brown trout, Salmo trutta Linnaeus, 1758 (see Arslan et al. 2012). Canola oil is particularly notable for its moderate n-3 content in the form of α-linolenic acid (18:3n-3), its low SFA content, and its high oleic acid (18:1n-9) content, which collectively confer a relatively favorable fatty acid balance (Gunstone 2011; Shepherd and Jackson 2013). Safflower oil, conversely, is characterized by an exceptionally high linoleic acid content (typically >70% of total fatty acids), low SFA, and negligible n-3 fatty acids, making it a useful tool for studying the specific effects of dietary n-6 PUFA enrichment on fish metabolism and tissue fatty acid composition (Altundağ et al. 2014).

Black Sea trout, Salmo labrax Pallas, 1814, is an anadromous salmonid species endemic to the Black Sea basin and its tributary rivers, including those along the northeastern coast of Turkey (Başçınar et al. 2010; Çakmak et al. 2018; Özel et al. 2018). This species has significant economic importance for Turkish aquaculture and commercial fishing and is one of the important rural livelihood sources in the Black Sea coastal region (Çakmak et al. 2018; Özel et al. 2018 , Özdemir et al. 2026). Despite this economic significance, the nutritional biology and dietary lipid requirements of Black Sea trout remain comparatively understudied relative to commercially dominant salmonid species such as Atlantic salmon and rainbow trout. To date, only a limited number of investigations have examined the effects of plant oil substitution on growth performance and tissue fatty acid composition in this species (Çakmak et al. 2018; Özel et al. 2018), and no published study has systematically evaluated the simultaneous use of both canola and safflower oils—individually and in binary and ternary combinations—as fish oil replacements in Black Sea trout diets.

The present study was therefore designed to evaluate the effects of replacing dietary fish oil with canola oil, safflower oil, and their blends on the growth performance, feed utilization efficiency, muscle proximate composition, and muscle and diet fatty acid profiles of juvenile Black Sea trout over a 90-day feeding trial. A secondary objective was to assess the economic implications of plant oil substitution through a feed cost analysis. The results of this study are intended to provide a scientific basis for the development of more sustainable and economically viable lipid nutrition strategies for Black Sea trout aquaculture.

Experimental animals, housing, and acclimation. Juvenile Black Sea trout (Salmo labrax) were obtained from a certified commercial fish farm in Rize, Turkey, and transported to the experimental facility at Sinop University, Turkey. The initial body weight (~30 g) reflects the size of juvenile fish available from the supplying commercial farm at the time of the trial, which were being reared toward portion size (approximately 250–300 g); no alternative size class was available from the source farm during this period. The 90-day trial duration is consistent with standard practice in comparative fish oil replacement feeding trials, which typically employ a 90-day period to allow sufficient time for dietary lipid source effects on growth, tissue composition, and organ indices to become measurable. Fish underwent a 30-day acclimation period in the experimental tanks under identical environmental conditions to those employed during the trial. During acclimation, fish were fed twice daily to apparent satiation using a commercial trout pellet diet containing approximately 44% crude protein and 18% crude lipid (NRC 2011). No fish showing signs of illness or exhibiting abnormal behavior were observed during the trial period.

After 24 h of fasting, fish were weighed individually to the nearest 0.1 g (initial mean body weight 30.22 ± 0.19 g). Fish were randomly distributed into 21 circular fiberglass tanks (300 L capacity, 25 fish per tank). Dietary treatments were randomly assigned to tank positions within the experimental system to minimize positional effects on growth and feed intake. Each treatment was designed with 3 replicates. Stocking density was maintained at levels reported to have no significant effect on growth performance in salmon; initial stocking density was approximately 2.5 kg · m⁻³ (25 fish per 300 L tank), increasing to approximately 6.8 kg · m⁻³ by the end of the trial. To perform the necessary analyses at the end of the experiment, 5 fish samples were taken from each tank.

Filtered groundwater was supplied to all tanks at a constant flow rate of 4.0 L · min⁻¹, with airstone diffusers for oxygen supplementation. Water quality parameters were monitored daily: temperature (15.7 ± 0.06°C) (Epinox DT-03 digital thermometer), dissolved oxygen (7.55 ± 0.15 mg · L⁻¹) (Oxyguard Handy Polaris brand oxygen meter), and pH (7.81 ± 0.02) (Thermo Scientific Orion brand pH meter), all within optimal ranges for Black Sea trout culture (Başçınar et al. 2010; Özel et al. 2018). Total ammonia nitrogen and nitrite were monitored daily using a colorimetric test kit and remained below 0.025 mg · L⁻¹ and 0.10 mg · L⁻¹, respectively. Photoperiod followed the natural light cycle.

Diet formulation and feeding protocol. Seven isonitrogenous and isolipidic experimental diets (approximately 45% crude protein and 18% crude lipid) were formulated to meet salmonid nutritional requirements (Tocher 2003; Rinchard et al. 2007). The diets differed only in terms of lipid source. Fish oil from fish meal (nearly 4%) was taken into account when calculating the basic feed formulation. The feed content is given in Table 1.

Three lipid sources were used: refined anchovy fish oil (FO), food-grade cold-pressed safflower oil (SO; Carthamus tinctorius), and food-grade refined canola oil (CO; Brassica napus) (Suppl. material 1: table SS1). Seven dietary lipid formulations were prepared: FO (100% fish oil), SO (100% safflower oil), CO (100% canola oil), FO-SO (50:50), FO-CO (50:50), SO-CO (50:50), and FO-SO-CO (equal thirds). Total lipid inclusion was held constant at 135 g · kg⁻¹. The oil addition rates in the blended diets were calculated to achieve the designated percentage of total added lipid from each source after accounting for the endogenous lipid contributed by fish meal (approximately 4% lipid, providing roughly 12 g lipid · kg⁻¹ diet). For example, in the FO-SO diet, the target was 50% of the added lipid from fish oil and 50% from safflower oil; after subtracting the fish meal lipid contribution, this required 46.95 g fish oil and 88.05 g safflower oil per kg diet. All blended diets were formulated using this same principle to ensure the intended proportional lipid contributions from each source. Although discrete graded substitution levels for a single plant oil (e.g., 25%, 50%, 75% CO) were not tested, the FO-SO and FO-CO diets provided an intermediate level of substitution (~65% plant oil on an oil-mass basis), allowing partial assessment of dose-dependent effects within the present seven-diet design.

Diets were manufactured using a standardized protocol: dry ingredients were mixed for 15 min, lipid blends were added incrementally and mixed for 10 min, distilled water (35% w/w) was added, and diets were pelleted through a 3.0 mm die, dried at 55°C for 8 h, vacuum-sealed, and stored at −20°C. Proximate analysis confirmed crude protein (45.54%–45.82%) and crude lipid (18.24%–18.42%) across diets (Table 1).

Fish were hand-fed to apparent satiation twice daily (09:00 and 15:00 h) for 90 days. Uneaten feed was collected 30 min after each feeding, dried (105°C, 24 h), and weighed to determine actual feed intake. Fish were fasted for 24 h prior to each weighing event.

Growth performance and biometric indices. All fish were batch-weighed at 15-day intervals to the nearest 0.1 g following a 24-h fast. Length measurements were recorded at the beginning and end of the trial. Mortalities were recorded daily and removed for correction of feed conversion calculations. Growth performance and feed utilization parameters were calculated using the following equations:

Weight gain (W_G) [%]

W_G = (W_F − W_I) · (100 · W_I)⁻¹

Specific growth rate (SGR) [% · day⁻¹]

SGR = (ln W_F − ln W_I) · (days · 100)⁻¹

Feed conversion ratio (FCR)

FCR = W_FO · W_G⁻¹

Thermal growth coefficient (TGC) (Jobling 2003)

TGC = [(WF^1/3 − WI1^/3) (T · t) ⁻¹·] 1000·

Fat deposition rate (FDR) [%· day⁻¹] (Richard et al. 2006)

FDR = (ln LC_F − ln LC_I) · (days · 100)⁻¹

Survival rate (SR) [%]

SR = 100 · N_F · N_I⁻¹

Protein efficiency ratio (PER)

PER = W_G · W_PI⁻¹

Hepatosomatic index (HSI) [%]

HSI = 100 · W_L · W_B^–1

Viscerosomatic index (VSI) [%]

VSI = 100 · W_V · W_B^–1

W_F is the final wet body weight [g]; W_I is the initial wet body weight [g]; W_FO is the weight of feed offered; T is the mean water temperature [°C]; t is the trial duration [days]; LC_F is the final whole-body lipid content; LC_I is the initial whole-body lipid content; N_F is the final fish number; N_I is the initial fish number; W_G is the weight gain [g]; W_PI is the protein intake [g]; W_L is the liver weight [g]; W_B is the body weight [g]; W_V is the viscera weight [g].

At the start of the experiment, 30 fish were sacrificed by rapid immersion in an overdose of buffered clove oil and frozen at −80°C for initial whole-body proximate composition (AOAC 1995). At trial conclusion, five fish per replicate tank (15 fish per dietary treatment) were sacrificed by the same method. Livers and viscera were dissected and weighed for HSI and VSI calculation. White dorsal muscle was excised, vacuum-sealed, and stored at −80°C.

Proximate and fatty acid composition analysis. Proximate compositions of diets, initial whole-body samples, and terminal muscle samples were determined in triplicate according to AOAC methods (AOAC 1995). Moisture was determined gravimetrically (105°C, 24 h). Crude protein (N × 6.25) was measured by the Kjeldahl method (Kjeltec 8400, FOSS Analytical, Hillerød, Denmark). Crude lipid was extracted with petroleum ether using a Soxhlet system (Soxtec 2043, FOSS Analytical, Hillerød, Denmark). Ash was determined by incineration at 550°C for 6 h. Gross energy calculated using standard conversion factors (Tocher 2003).

Fatty acid analyses were conducted on the seven experimental diets, dietary oil sources (fish oil, canola oil, and safflower oil), and muscle samples obtained from fish at the beginning and termination of the feeding trial. Analyses were performed by the TÜBİTAK Marmara Research Center (MAM, Gebze, Türkiye) through a contracted analytical service. Fatty acid composition was determined in the extracted lipid fraction following the IUPAC IID19 method, and individual fatty acids were quantified and expressed as percentages of total fatty acids.

Economic analysis. Feed cost analysis was conducted for all seven experimental diets using indicative ingredient prices (USD · kg⁻¹): fish meal (1.80), soybean meal (0.55), corn gluten meal (0.65), sunflower meal (0.35), wheat flour (0.40), fish oil (2.20), safflower oil (1.15), canola oil (1.05), vitamin premix (5.00), and mineral premix (3.00). These values represent approximate 2024 global market averages compiled from general commodity and industry price trends reported in Tacon et al. (2011), Shepherd and Jackson (2013), and IFFO (2024) rather than direct supplier quotations, and are intended to illustrate the relative cost structure of the experimental diets rather than to provide region-specific or invoice-level costing; readers applying this analysis to a specific production context should substitute locally verified ingredient prices. Feed cost per kilogram of diet (FCD) was calculated as the sum of ingredient inclusion levels multiplied by unit prices, divided by 1000. Feed cost per kilogram of fish produced (FCPF) was calculated (Hurwitz et al. 2023):

FCPF = FCD · FCR

Relative cost saving (RCS) [%] was calculated as:

RCS = (FCD_FO – FCD_treatment) · (FCD_FO · 100)^–1

where FCD_FO is the feed cost per kilogram of the fish oil (FO) diet, and FCD_treatment is the feed cost per kilogram of each experimental (treatment) diet.

Statistical analysis. All data are expressed as mean ± SEM (standard error of the mean). Normality was assessed using the Shapiro–Wilk test and homogeneity of variances using Levene’s test. Treatment effects were evaluated by one-way ANOVA, the Kruskal–Wallis test (when non-normal). Post-hoc comparisons were conducted using Tukey’s HSD or Tamhane’s T2 test. Significance was set at P = 0.05. All statistical computations were performed using IBM SPSS Statistics, version 21.0 (IBM Corp. 2012, Armonk, New York, USA).

Ethical Statement. All experimental procedures were approved by the Sinop University Animal Experiments Local Ethics Committee (decision number 2015/11, 13 April 2015). Animal welfare principles of the 3Rs (Replacement, Reduction, Refinement) were applied throughout the study.

Growth performance, feed utilization, and condition indices. All seven experimental diets were readily consumed by Black Sea trout throughout the 90-day feeding trial, with no feed refusal or aberrant feeding behavior observed in any dietary group.

One-way ANOVA revealed no statistically significant differences (P > 0.05) among dietary treatments in final mean body weight, weight gain (W_G), specific growth rate (SGR), feed conversion ratio (FCR), thermal growth coefficient (TGC), survival rate (SR), and protein efficiency ratio (PER) (Table 2). The weight gain ranged from 163.69% (SO-CO) to 166.95% (FO), and SGR ranged from 1.10% · day⁻¹ (FO-SO) to 1.11% · day⁻¹ (SO). FCR ranged from 1.11 (CO) to 1.17 (FO-CO). TGC ranged from 0.86 to 0.87, SR from 90.00% to 96.00%, and PER from 1.95 (FO, FO-SO) to 2.20 (SO-CO) (P > 0.05 for all).

Fat deposition rate (FDR) differed significantly among dietary groups (P < 0.05), ranging from 0.47% · day⁻¹ (FO) to 0.61% · day⁻¹ (FO-CO). Viscerosomatic index (VSI) and hepatosomatic index (HSI) also differed significantly among groups (P < 0.05). Terminal VSI ranged from 9.75% (CO) to 12.94% (SO-CO), compared with an initial value of 9.16%. HSI ranged from 1.63% (CO) to 2.16% (SO-CO); the SO and CO groups recorded significantly lower HSI (1.75% and 1.63%, respectively) than the FO control (1.94%) and the SO-CO blend (2.16%) (Fig. 1).

Muscle proximate composition. Muscle protein and ash contents did not differ significantly among dietary treatments (P > 0.05; Table 3). Muscle moisture and lipid contents differed significantly among treatments (P < 0.05). The highest muscle lipid content was recorded in fish fed the FO-CO diet (5.79 ± 0.03%) and the FO-SO diet (5.74 ± 0.09%), and the lowest in fish fed the FO control diet (5.12 ± 0.11%) and the SO-CO diet (5.16 ± 0.08%).

Fatty acid composition of experimental diets. The fatty acid profiles of the seven experimental diets are presented in Table 4 and visualized in Fig. 2. Total saturated fatty acids (ΣSFA) ranged from 11.80% (CO) to 37.94% (FO), with palmitic acid (16:0) the dominant contributor (7.39% in CO to 22.77% in FO). Total monounsaturated fatty acids (ΣMUFA) ranged from 23.69% (SO) to 52.25% (CO), driven by oleic acid (18:1n-9; 17.98% in FO to 49.94% in CO). Total polyunsaturated fatty acids (ΣPUFA) ranged from 25.25% (FO) to 60.19% (SO). Linoleic acid (LA; 18:2n-6) ranged from 7.39% (FO) to 56.28% (SO), and α-linolenic acid (LNA; 18:3n-3) ranged from 0.38% (SO) to 7.30% (CO). EPA (20:5n-3) ranged from 0.82% (FO-SO-CO) to 6.33% (FO), and DHA (22:6n-3) ranged from 1.13% (FO-SO-CO) to 7.47% (FO); both EPA and DHA were detectable, at levels of 1.07%–1.17% and 2.05%–2.16%, respectively, even in the 100% plant oil diets (SO, CO). Total n-3 fatty acids (Σn-3) were highest in the FO diet (15.66%) and lowest in the SO diet (3.58%); total n-6 fatty acids (Σn-6) were highest in the SO diet (56.39%) and lowest in the FO diet (8.15%). The dietary n-3/n-6 ratio ranged from 0.06 (SO) to 1.92 (FO) (Fig. 3).

Fatty acid composition of muscle tissue. The fatty acid profiles of white dorsal muscle are presented in Table 5 (Fig. 4). Total muscle SFA ranged from 15.40% (CO) to 23.51% (FO-SO). Total muscle MUFA ranged from 29.31% (FO-SO) to 52.83% (CO), with oleic acid ranging from 24.95% (FO-SO) to 48.57% (CO). Total muscle PUFA ranged from 22.69% (FO-CO) to 41.82% (SO) (Suppl. material 1: table SS2). Muscle LA ranged from 11.49% (FO) to 36.32% (SO). Muscle EPA ranged from 0.12% (SO) to 2.26% (FO), and muscle DHA ranged from 0.67% (SO) to 7.10% (FO). Total muscle n-3 fatty acids ranged from 1.41% (SO) to 11.39% (FO), and total muscle n-6 fatty acids ranged from 12.15% (FO) to 38.48% (SO). The muscle n-3/n-6 ratio ranged from 0.04 (SO) to 0.94 (FO) (Fig. 3).

Economic analysis. Feed cost per kilogram of diet (FCD), feed cost per kilogram of fish produced (FCPF), and relative cost saving (RCS) are presented in Table 6 (Fig. 5). The highest FCD was recorded for the FO diet (1.153 USD · kg⁻¹) and the lowest for the CO diet (0.998 USD · kg⁻¹), followed by SO (1.012 USD · kg⁻¹); partial replacement diets showed intermediate FCD values (1.005–1.061 USD · kg⁻¹). The lowest FCPF was recorded for the CO diet (1.108 USD · kg⁻¹ per fish produced. and the highest for the FO diet (1.292 USD · kg⁻¹ per fish produced). Relative cost savings compared with the FO control were positive for all plant oil-containing diets, ranging from +8.02% (FO-SO) to +13.46% (CO).

A recent meta-analysis of 328 studies confirmed that the effects of plant oil substitution on growth and lipid metabolism are species-specific and dose-dependent across major cultured fish (Qian et al. 2024). Numerous studies have demonstrated the potential to replace significant amounts of dietary fish oil with plant oils (canola, safflower, soybean, sunflower, linseed, corn, sesame, peanut, hazelnut, and olive oils) in the diets of various fish species such as brown trout (Arslan et al. 2012), Caspian trout (Sajedkhanian et al. 2021), bull trout (Twibell et al. 2016), brook charr (Dupont-Cyr et al. 2022), Arctic charr (Dupont-Cyr et al. 2022), rainbow trout (Acar and Turker 2018; Yıldız et al. 2018), Atlantic salmon (Torstensen et al. 2004; Davis and Devine 2023) gilthead sea bream (Sánchez-Moya et al. 2020), and European sea bass (Verge-Mèrida et al. 2022), without negatively affecting the growth performance or feed conversion efficiency. More recent work continues to support this pattern: Song et al. (2024) reported no significant differences in growth rate, condition factor, or yield when fish oil was fully replaced by canola oil in triploid rainbow trout, while Berge et al. (2023) directly compared EPA and DHA utilization efficiency between Atlantic salmon and rainbow trout fed rapeseed oil-substituted diets, and Davis and Devine (2023) reviewed canola oil’s suitability as a long-chain omega-3 source across the Atlantic salmon life cycle. The results of this study are consistent with these findings. In this study, replacing fish oil with varying ratios of canola and safflower oils did not negatively affect the growth performance of Black Sea trout. Arslan et al. (2012) reported that the highest growth performance in juvenile brown trout was achieved with a diet containing a blend of linseed and soybean oils. Similarly, our study found that safflower and canola oils can be used at levels up to 100% in Black Sea trout diets without negatively affecting growth performance or feed conversion. Comparable results have also been observed in studies with other fish species. Dernekbaşı et al. (2015) replaced up to 50% of fish oil with safflower oil and up to 66% with a blend of safflower and canola oils in rainbow trout diets, observing no significant effect on growth. Similarly, Blanchard et al. (2008) replaced 50% of cod liver oil with safflower oil in the diet of Eurasian perch without noting any negative effects on growth. Izquierdo et al. (2003) reported that replacing up to 60% of fish oil with various plant oil sources (such as soybean, rapeseed, and linseed oils or a mixture of these) did not compromise growth performance, feed utilization efficiency, or flesh quality in different fish species including sea bream and European sea bass. These findings align with the results of the present study regarding fish growth.

Black Sea trout (Salmo labrax) is an anadromous salmonid species that naturally migrates between freshwater rivers and the marine Black Sea environment, where it has access to marine prey rich in n-3 long-chain polyunsaturated fatty acids (Turchini et al. 2009; Çakmak et al. 2018). This evolutionary life history may have shaped its lipid metabolism and essential fatty acid requirements differently from purely freshwater species (such as brown trout) or strictly marine species (such as gilthead sea bream). Juvenile Black Sea trout, like other salmonids, are believed to have a limited capacity for endogenous LC-PUFA biosynthesis from 18-carbon precursors, but direct evidence for this species is lacking (Tocher 2003; Tocher et al. 2006). This capacity is governed by the fatty acyl desaturase (fads2) and elongase (elovl) gene repertoire, which in salmonids has been shaped by a genome-wide duplication event (4R) distinct from that of most other teleosts, potentially conferring a broader enzymatic toolkit for LC-PUFA biosynthesis than in strictly marine species (Monroig et al. 2022).

The presently reported results suggest that despite this anadromous life history, juvenile Black Sea trout show similar patterns of dietary fatty acid deposition and tissue lipid composition as other salmonids. The observation that muscle fatty acid profiles closely tracked dietary composition—with n-3 LC-PUFA levels declining as plant oil inclusion increased—indicates that this species, like Atlantic salmon and rainbow trout, relies heavily on dietary supply to meet its essential fatty acid requirements (Bell et al. 2001; Torstensen et al. 2004). However, the measurable EPA and DHA levels observed in muscle tissue of 100% plant oil-fed fish, and the significantly higher levels in canola oil-fed compared to safflower oil-fed fish, raise the possibility that S. labrax possesses some capacity to elongate and desaturate 18:3n-3 to longer-chain n-3 PUFA. This is a hypothesis generated by the present data rather than a previously documented capacity in this species; it is nonetheless consistent with reports that other salmonids fed vegetable-oil-based diets exhibit increased expression of hepatic LC-PUFA biosynthesis genes (Betancor et al. 2016), and it warrants direct investigation using stable isotope tracer studies, as discussed below.

In the presently reported study, replacing fish oil with varying levels of safflower and canola oils had no significant effect on the FCR, which ranged from 1.11 to 1.17. These FCR values are consistent with those reported by Başçınar et al. (2010) for the growth performance of brook trout and Black Sea trout under cultured conditions. Özel et al. (2018) investigated the effects of soybean oil and linseed oil mixtures (67%–33%, 50%–50%, 33%–67%) on the growth performance and fat metabolism of Black Sea trout and reported FCR values ranging from 1.02 to 1.30, which are comparable to those observed in the present study. Similarly, the FCR values observed in this study are comparable to those reported by Arslan et al. (2012) in their research on replacing fish oil with various plant oils in brown trout diets. These findings suggest that canola oil and safflower oil can completely replace fish oil in the diet of Black Sea trout without adversely affecting growth performance and feed conversion efficiency.

It is also worth noting that gross energy content was nearly identical across all seven diets (22.24–22.35 kJ · g⁻¹; Table 1), despite their markedly different lipid sources. This near-isoenergetic formulation indicates that the absence of treatment effects on growth, feed conversion, and survival cannot be attributed to differences in dietary energy density, and that any differences in organ indices or fatty acid composition reported below are more plausibly linked to the specific fatty acid profile of each lipid source rather than to a confounding difference in total energy supply.

Growth performance did not differ among dietary treatments, yet organ indices and muscle fatty acid composition did, and this dissociation is the central finding of the present study: it shows that growth alone cannot be relied upon to judge whether a lipid source is an adequate fish oil substitute in Black Sea trout. Canola and safflower oil, tested individually and in binary and ternary combination, were each able to fully replace added fish oil without a growth penalty, but the oil blends did not always produce intermediate physiological responses, particularly with respect to hepatic lipid metabolism. This has a direct bearing on commercial feed formulation, where blends rather than single oils are the norm.

Three findings deserve particular attention. First, fat deposition rate, viscerosomatic index, and hepatosomatic index diverged significantly among treatments despite uniform weight gain, feed conversion, and survival, indicating that dietary lipid source altered the partitioning of absorbed lipid among somatic, visceral, and hepatic compartments without altering whole-body growth trajectory. Second, hepatosomatic index was significantly higher in fish fed the SO-CO blend (2.16%) than in fish that were fed the single plant oils, CO (1.63%) and SO (1.75%), while the fish oil control (1.94%) and other blend groups (FO-SO: 1.82%; FO-CO: 1.95%; FO-SO-CO: 1.81%) were statistically intermediate and did not differ significantly from either the single oils or the SO-CO blend. This pattern is not explained by total plant oil inclusion alone, since SO-CO itself contained no fish oil, yet produced the numerically highest HSI among all treatments. Third, the dietary and muscle n-3/n-6 ratios collapsed by more than an order of magnitude in the SO group relative to FO (0.06 versus 1.92 in the diet; 0.04 versus 0.94 in muscle), a shift considerably larger than the corresponding shift in the CO group, even though both oils achieved 100% substitution of added fish oil.

Our results agree with the wider literature in showing that growth performance and feed conversion in salmonids are generally unaffected by partial or complete replacement of fish oil with canola or safflower oil (Izquierdo et al. 2003; Blanchard et al. 2008; Arslan et al. 2012; Dernekbaşı et al. 2015). Where the present findings go beyond this established pattern is in the organ-index and fatty-acid-ratio results described above, neither of which has previously been documented for Black Sea trout; the HSI pattern observed in the SO-CO group specifically has not been reported for any safflower-canola combination in any salmonid.

Non-additive blend effects. The present findings reveal three observations that challenge the assumption that blended diets behave as simple linear intermediates between their single-oil components. First, the hepatosomatic index in the SO-CO group (2.16%) was significantly higher than in either the CO (1.63%) or SO (1.75%) single-oil groups, and this elevation was not explained by total plant oil content, fish oil presence, or dietary energy density (Table 2). Second, muscle ΣSFA in the FO-SO group (23.51%) exceeded both its FO (22.18%) and SO (21.30%) parents, while muscle ΣMUFA in the FO-CO group (47.06%) fell short of the proportional mixing prediction (50.5%) derived from its FO (45.99%) and CO (52.83%) parents. Third, these deviations occurred despite dietary fatty acid class values in the blended diets being closely predicted by simple linear mixing of the parent oils, confirming that the non-additivity arises at the level of tissue lipid metabolism rather than at feed formulation.

The elevated hepatosomatic index in the SO-CO group relative to either single-oil diet cannot be explained mechanistically with the present dataset, since no hepatic gene expression, enzyme activity, or lipid transport protein data were collected in this study. As a direction for future work only, we note that the intermediate n-6/n-3 ratio of the SO-CO blend (dietary n-3/n-6 = 0.16) could plausibly influence hepatic lipogenic gene expression (e.g., srebp1c, fas, acc) and/or hepatic lipid export capacity (e.g., apoB100, mtp), both of which are known in salmonids to respond to dietary fatty acid composition (Bell et al. 2001; Bell et al. 2002; Tocher 2003). This is an untested hypothesis, not a conclusion drawn from the present data. Non-additive hepatic responses to blended vegetable oils have been reported in other salmonids (Tocher et al. 2001; Tocher et al. 2003), which lends the hypothesis biological plausibility but does not confirm it for S. labrax. Direct testing would require hepatic gene expression analysis in fish fed the FO, SO, CO, and SO-CO diets specifically.

The non-additive deviations in muscle fatty acid class values observed in the blended groups further suggest that competition between fatty acid classes for incorporation into tissue lipids is not simply proportional to dietary supply. The elevated ΣSFA in FO-SO relative to both parents, and the reduced ΣMUFA in FO-CO relative to its proportional prediction, imply that the presence of two lipid sources simultaneously may alter the relative rates of fatty acid incorporation, oxidation, or selective retention at the tissue level. This observation aligns with the concept of “fatty acid sparing” described in other salmonids (Turchini et al. 2011), where the presence of certain fatty acids can influence the metabolic fate of others, but extends this concept to blended oils.

The lower hepatosomatic index in the CO and SO groups relative to FO is likely attributable to reduced hepatic lipid storage in fish fed diets lacking the saturated fatty acid load of fish oil, consistent with reports that high dietary SFA promotes hepatic lipid accumulation in salmonids whereas high MUFA or PUFA intake favors lipid export or oxidation over storage (Bell et al. 2001; Bell et al. 2002). This mechanism, however, would predict a monotonic decline in HSI as plant oil inclusion increases, which is not consistent with the full pattern observed: although the SO-CO blend recorded the numerically highest mean HSI (2.16%), post-hoc comparisons showed this value differed significantly only from the CO and SO groups themselves (1.63% and 1.75%, respectively), while FO (1.94%), FO-SO (1.82%), FO-CO (1.95%), and FO-SO-CO (1.81%) all overlapped statistically with both the SO-CO and the single-plant-oil groups (Table 2). The clearest statistically supported contrast is therefore that the two single-plant-oil diets produced significantly lower HSI than their corresponding 50:50 blend with the other plant oil, while the fish oil control and other blends occupied an intermediate position that did not differ significantly from either extreme.

Plant oils typically contain higher levels of n-6 PUFAs than fish oil, which is primarily rich in n-3 PUFAs, such as EPA and DHA, along with LCMUFAs. In recent years, plant oils have been increasingly recognized as alternative lipid sources in fish diets. Consequently, research in aquaculture has intensified to replace fish oil with various plant-derived ingredients. Turchini et al. (2019) reported that certain combinations of lipid sources may be more effective than fish oil in terms of n-3 LC-PUFA bioavailability and efficiency in different fish species. In this study, the highest amount of SFA was observed in the FO diet (37.94%), which contained 100% fish oil. The higher SFA content in fish oil, compared to safflower and canola oils, resulted in greater total amount of SFA in the experimental diets. The diets with the lowest total SFA content were those containing only safflower and canola oils (13.82% in the SO diet, 11.80% in the CO diet, and 13.96% in the SO-CO diet). The highest muscle SFA values were recorded in the FO-SO (23.51%), FO-CO (22.73%), and FO (22.18%) groups, broadly reflecting the higher dietary SFA load supplied by fish oil in these treatments. This pattern was not, however, uniform across every fish-oil-containing diet: the FO-SO-CO group, which also included a fish oil component (17.6 g · kg⁻¹), recorded a comparatively low muscle SFA value (17.09%), lower than several diets containing no fish oil at all (SO: 21.30%; SO-CO: 18.89%). This indicates that the proportion and combination of plant oils blended with fish oil, rather than the simple presence or absence of fish oil, determines muscle SFA outcome once more than one plant oil is involved. Similar results have been reported for several fish species in other studies (Bell et al. 2001, 2002, 2003; Blanchard et al. 2008; Özel et al. 2018), where certain plant oils (e.g., canola, soybean, linseed oil) were used as replacements for fish oil. Özel et al. (2018) reported that the highest SFA content (22.93%) in the feed was observed in the fish oil-containing feed. Accordingly, SFA levels in fish muscle were higher in fish fed the fish oil-containing feed, whereas those fed the soybean and linseed oil mixture had lower SFA levels. Similarly, Dernekbaşı et al. (2015) found that the highest total SFA level (30.98%) was in the feed containing 100% fish oil, with total SFA levels decreasing as the proportion of safflower oil increased. Our study, along with others, suggests that the total SFA content of the lipid blend, rather than the presence of fish oil per se, determines total SFA in the feed and muscle.

In this study, the highest MUFA content (52.25%) was observed in the CO diet, which contained 100% canola oil, while the lowest content (23.69%) was found in the diet containing 100% safflower oil. Among the monounsaturated fatty acids, oleic acid was the most dominant, reaching 49.94% of total fatty acids in the CO diet. This is consistent with the inherently high oleic acid content of neat canola oil, commonly reported at around 60%–62% of total fatty acids in the oil itself (Gunstone 2011); the somewhat lower value observed here in the formulated CO diet (49.94%) reflects the dilution of canola oil’s fatty acid profile by the other, oleic-acid-poor basal ingredients common to all experimental diets. The dietary composition influenced the fatty acid profile of the fish muscle, with higher oleic acid (48.57%) and MUFA (52.83%) values observed in fish fed the 100% canola oil diet, while lower values were found in fish fed safflower oil-containing diets. Similarly, Yıldız et al. (2018) reported that using cottonseed oil and canola oil as replacements for fish oil in rainbow trout feed resulted in the highest oleic acid (49.9%) and MUFA (56.1%) levels in the canola oil diet. By the end of their study, the highest oleic acid (45.7%) and MUFA (53.7%) levels in the fish fillet were observed in fish fed the 100% canola oil diet. Similar results have been reported in other studies using canola oil (Twibell et al. 2016; Mock et al. 2021; Azcuy et al. 2025). It was observed that as the proportion of plant oils replaced fish oil in the experimental diets, the total amount of n-6 PUFA increased, while the total amount of n-3 PUFA decreased. Due to its high linoleic acid content, feeds containing safflower oil had higher total n-6 PUFA levels. Dernekbaşı et al. (2015) found that the highest total PUFA and n-6 PUFA levels in rainbow trout feed were achieved by replacing 50% of fish oil with safflower oil. Similar findings have been reported in other studies using safflower oil in the diets (Altundağ et al. 2014; Ustaoğlu Tırıl et al. 2016).

Linoleic acid is the predominant fatty acid in the n-6 fatty acid group. In this study, the amount of linoleic acid was higher in fish muscle fed diets containing safflower oil. The highest level of linoleic acid was observed in the group fed the diet containing 100% safflower oil (36.32%), while the lowest level was found in the group fed the 100% fish oil diet (11.49%). Özel et al. (2018) reported that the highest linoleic acid content in Black Sea trout muscle (26.96%) was observed in the group fed a diet containing 67%–33% soybean and linseed oils. Similarly, Dernekbaşı et al. (2015) and Dernekbaşı and Karataş (2020) determined that the linoleic acid and total n-6 levels in rainbow trout muscle increased as the proportion of safflower oil in the diets increased. Many studies have reported that as the amount of plant oil (e.g., soybean oil, linseed oil, hazelnut oil, canola oil, palm oil, cotton seed oil, etc.) used to replace fish oil increases, the linoleic acid and total n-6 content in both the feed and fish meat also increase (Arslan et al. 2012; Dernekbaşı et al. 2015; Yıldız et al. 2018; Sajedkhanian et al. 2021). These results are consistent with our findings.

This dose-dependent relationship between dietary and tissue n-3 LC-PUFA content has recently been confirmed quantitatively across cultured fish species through meta-analysis (Wu et al. 2024). EPA and DHA are essential fatty acids for fish survival and growth. Fish feeds containing fish oil are typically rich in these fatty acids, which are also found in the tissues of fish fed such diets. In contrast, fish fed diets containing plant oils instead of fish oil have lower levels of EPA and DHA, as reported in several studies (Arslan et al. 2012; Dernekbaşı et al. 2015; Ustaoğlu Tırıl et al. 2016; Yıldız et al. 2018; Sajedkhanian et al. 2021). Numerous studies on the replacement of fish oil have demonstrated that various plant oils, including soybean, rapeseed, linseed, corn, and palm oils, can serve as substitutes. However, when plant oil inclusion exceeds 50% of the diet, a significant accumulation of 18:2n-6 and a reduction in EPA and DHA levels in fish tissue occurs (Torstensen et al. 2000; Bell et al. 2001; Bell et al. 2003; Torstensen et al. 2004). Similar results were obtained in this study, where fish feeds containing fish oil had higher levels of EPA and DHA, while these fatty acids decreased as the proportion of plant oil in the feed increased. This pattern has also been confirmed very recently in rainbow trout, where canola oil-fed fish showed elevated fillet MUFA and n-6 PUFA at the expense of EPA and DHA relative to fish oil-fed fish (Azcuy et al. 2025). In two different studies on rainbow trout using safflower oil and canola oil, it was reported that as the amount of safflower and canola oil increased, EPA and DHA levels in fish tissue decreased, with the lowest levels observed in fish fed the diet containing the highest proportion of safflower oil (Blanchard et al. 2008; Dernekbaşı et al. 2021). A study on Black Sea trout revealed that fish fed diets containing vegetable oils exhibited the lowest concentrations of EPA and DHA in muscle tissue, whereas the highest levels were detected in those fed a fish oil-based diet (Çakmak et al. 2018). The findings of this study are consistent with those of other studies that used plant oils (Arslan et al. 2012; Çakmak et al. 2018; Yıldız et al. 2018; Dernekbaşı et al. 2021; Sajedkhanian et al. 2021).

This appears to be the first study in Black Sea trout to test canola and safflower oil simultaneously, both individually and in binary and ternary combination, and the first to pair the resulting fatty acid data with a feed-cost analysis for this species. Prior work on alternative lipid sources in Black Sea trout has been limited, and neither the organ-index pattern nor the magnitude of the n-3/n-6 shifts reported here has previously been documented for this species, consistent with how little is known about its dietary lipid requirements relative to Atlantic salmon and rainbow trout.

The fatty acid profiles of the partial-substitution diets (FO-SO, FO-CO, SO-CO, and FO-SO-CO) provide additional resolution on how blending modulates the dietary and muscle fatty acid supply, a dimension not addressed by the single-oil comparisons discussed above. It should be noted that the binary blends were not formulated on a 50:50 weight basis but reflect the oil quantities required to supply 50% of total dietary lipid from each source after accounting for the fixed fish meal lipid contribution (FO-SO: 46.95 g fish oil and 88.05 g safflower oil per kg diet; FO-CO: 46.95 g fish oil and 88.05 g canola oil per kg diet). When this actual oil-mass ratio (approximately 35:65, fish oil to plant oil) is used to predict dietary fatty acid values by weighted linear mixing of the parent single-oil diets, the predictions match the observed values closely: dietary linoleic acid in FO-SO is predicted at 39.28% against an observed 39.30%, and dietary oleic acid in FO-CO is predicted at 38.83% against an observed 38.48%. This close agreement confirms that dietary fatty acid composition in the blended diets is explained almost entirely by simple proportional mixing of the parent oils, with no evidence of any interactive or non-additive effect at the level of feed formulation itself.

In contrast to the close agreement found at the level of dietary fatty acid classes, muscle fatty acid composition in the blended-oil groups deviated more noticeably from the values predicted by simple proportional mixing of the parent single-oil groups. Using the same oil-mass weighting applied above, muscle ΣSFA in the FO-SO group is predicted at 21.6% from its FO (22.18%) and SO (21.30%) parents, yet the observed value (23.51%) exceeds both parents, rather than falling between them; conversely, muscle ΣMUFA in the FO-CO group is predicted at 50.5% from its FO (45.99%) and CO (52.83%) parents, yet the observed value (47.06%) falls short of this prediction. These two deviations, in opposite directions, indicate that muscle-level handling of blended dietary fatty acids does not simply mirror the proportional mixing seen in the diet itself, reinforcing the inference drawn above from the hepatosomatic index that blended-oil diets can elicit tissue-level responses not predictable from their single-oil components considered separately. The n-3/n-6 ratios of the blended diets further illustrate the dominant role of safflower oil in driving n-3 depletion. The FO-CO blend (dietary n-3/n-6 = 0.92; muscle = 0.37) maintained a ratio more than three-fold higher than FO-SO (dietary 0.26; muscle 0.15), despite both diets replacing the same 65% of dietary lipid with a single plant oil. This difference arises because canola oil contributes α-linolenic acid (7.30% of total fatty acids) in addition to oleic acid, whereas safflower oil contributes almost exclusively linoleic acid (56.28%). The ternary blend (FO-SO-CO; dietary n-3/n-6 = 0.15; muscle = 0.22) underscores this pattern: its dietary ratio sits closer to the safflower-dominated end of the gradient than a three-way average would predict. Together with the muscle SFA and MUFA deviations described above, this indicates that the safflower oil fraction of a blend, specifically, rather than the total plant oil fraction, is the principal determinant of n-3/n-6 depletion in Black Sea trout muscle.

The contrast in dietary and muscle n-3/n-6 ratios between the SO and CO groups is the result with the clearest practical relevance for product quality, and it follows directly and quantitatively from the divergent fatty acid composition of the two oils rather than from any difference in fish physiology: canola oil retains a meaningful proportion of α-linolenic acid (7.30% of total dietary fatty acids) whereas safflower oil does not (0.38%), and this single compositional difference, propagated through diet into muscle, is sufficient to explain why CO-fed fish maintained a muscle n-3/n-6 ratio roughly eight-fold higher than SO-fed fish despite both diets containing zero added fish oil. This is consistent with prior demonstrations that muscle fatty acid composition in salmonids tracks dietary fatty acid supply closely (Torstensen et al. 2000, 2004; Bell et al. 2001), but the present comparison isolates the specific contribution of the α-linolenic acid difference between two plant oils rather than the more commonly studied fish oil-versus-plant oil contrast.

The observed changes in muscle n-3/n-6 ratio have direct implications for human nutrition and product marketing. The World Health Organization recommends a dietary n-3/n-6 ratio of approximately 0.2 or higher for optimal cardiovascular health and anti-inflammatory balance (WHO 2008; Simopoulos 2016). The physiological basis for this recommendation lies in the shared enzymatic pathways of n-3 and n-6 fatty acids: EPA and DHA are precursors of anti-inflammatory and pro-resolving eicosanoids and docosanoids, whereas linoleic acid-derived arachidonic acid gives rise to more pro-inflammatory signaling molecules, so a low dietary n-3/n-6 ratio is thought to shift this balance toward a pro-inflammatory state (Calder 2015; Simopoulos 2016). In this study, the FO group (muscle n-3/n-6 = 0.94), the CO group (0.32), and the FO-CO group (0.37) all met or exceeded this threshold, whereas the SO group (0.04) and the remaining blended groups (FO-SO: 0.15; SO-CO: 0.10; FO-SO-CO: 0.22) fell below or only marginally met this value. Notably, every diet containing canola oil—whether as the sole lipid source (CO) or in combination with fish oil (FO-CO)—satisfied the WHO threshold, whereas no diet containing safflower oil without fish oil did so. This suggests that long-term feeding of safflower oil, particularly as a sole substitute, may compromise the functional food status of Black Sea trout fillets, potentially reducing consumer appeal in markets that value omega-3 content (Sprague et al. 2016; Turchini et al. 2019), and further reinforces canola oil as the more nutritionally reliable plant-based option among those tested. From a product marketing and consumer acceptance standpoint, muscle n-3/n-6 ratio and absolute EPA+DHA content are increasingly used as label claims and premium-positioning criteria for farmed fish in export markets (Sprague et al. 2016); regulatory frameworks such as the EU’s nutrition and health claims regulation (EC 1924/2006) permit “source of omega-3” or “high omega-3” labeling only above defined EPA+DHA thresholds, so a fillet’s absolute EPA+DHA content, not only its n-3/n-6 ratio, can determine whether it qualifies for such marketing claims in key export destinations; a fillet failing to meet the WHO-referenced threshold may therefore face reduced marketability irrespective of its acceptable growth and feed conversion performance, underscoring that fillet nutritional quality and growth performance should be evaluated as independent, not interchangeable, criteria when selecting a fish oil substitute. It is worth noting, however, that this nutritional trade-off may be partly offset by a quality trade-off in the opposite direction: because EPA and DHA are more susceptible to lipid peroxidation than the MUFA- and n-6-enriched fatty acid profile of plant-oil-fed fish, fillets from the CO and SO groups may, in principle, exhibit greater oxidative stability during storage and processing, even as their omega-3 content declines. This possibility was not directly assessed in the present study (see Limitations) and would need to be confirmed through fillet shelf-life and peroxide value analyses.

From a commercial perspective, this nutritional trade-off should be weighed against the cost savings when selecting lipid sources for grow-out diets. While safflower oil offers a 12.29% estimated cost saving, the resulting product would have substantially lower nutritional value for human consumers compared to fish oil- or canola oil-fed fish. In contrast, canola oil provides a 13.46% estimated cost saving while maintaining a muscle n-3/n-6 ratio (0.32) that meets WHO recommendations and is eight-fold higher than safflower oil-fed fish. This distinction is particularly relevant for premium markets where rich in omega-3 claims are used as marketing tools (EFSA 2012; FAO 2022).

For producers targeting such markets, we recommend a pre-harvest finishing strategy using fish oil or high-n-3 feed sources to restore muscle EPA and DHA levels. Based on the depletion patterns observed in this 90-day study and comparable findings in Atlantic salmon and rainbow trout (Bell et al. 2003; Torstensen et al. 2005; Yıldız et al. 2018), a finishing period of 4–6 weeks appears sufficient to substantially restore muscle n-3 LC-PUFA levels, though this specific timeline requires direct validation in Black Sea trout. This two-phase feeding strategy—plant oil grow-out followed by fish oil finishing—may offer an optimal balance between production cost savings during the grow-out phase and product quality at harvest. From an operational standpoint, this strategy requires no specialized infrastructure beyond a temporary switch back to a fish-oil-based diet in the final weeks before harvest, making it readily implementable within existing commercial production schedules; the main practical trade-off is the partial loss of the feed-cost savings achieved during grow-out, concentrated into a short pre-harvest window rather than sustained throughout the production cycle.

The 100% CO and 100% SO diets, despite containing no added fish oil, still contained measurable EPA (1.17% and 1.07% of total fatty acids, respectively) and DHA (2.16% and 2.05%). Because fish meal was held constant at 300 g kg⁻¹ across all seven diets, the most likely explanation is that this residual EPA and DHA come from the fish meal’s own lipid fraction rather than from the labelled oil component. Whether the EPA and DHA found in the muscle of CO- and SO-fed fish reflects passive accumulation of this fishmeal-derived fraction alone, or some degree of endogenous elongation from the plant oils themselves, cannot be resolved with the present data. However, the dietary-to-muscle ALA/EPA+DHA transfer pattern—with CO-fed fish maintaining substantially higher muscle EPA (0.38%) and DHA (2.12%) than SO-fed fish (0.12% and 0.67%, respectively)—suggests that endogenous conversion may be occurring. Distinguishing between passive retention and active biosynthesis would require stable isotope tracer studies—a logical next step to characterize the LC-PUFA biosynthetic capacity of S. labrax, about which virtually nothing is currently known.

From an economic perspective, the relative cost savings estimated for complete substitution (+13.46% for CO, +12.29% for SO) are consistent with the general price advantage of plant oils over fish oil reported in the wider literature, reflecting the volatility and finite supply of forage-fish-derived oil relative to the large-scale, geographically diversified production of canola and safflower oil (Tacon et al. 2011; Shepherd and Jackson 2013). Beyond the point estimates of cost saving reported here, it is also relevant to note that fish oil prices have historically been considerably more volatile than canola or safflower oil prices: fish oil supply is concentrated in a small number of forage-fishery regions and is therefore highly sensitive to single-region shocks such as annual catch-quota adjustments, whereas canola and safflower oil are produced from geographically diversified, large-scale agricultural sources with comparatively more gradual, seasonally driven price movements (Shepherd and Jackson 2013; IFFO 2024). This structural difference in price stability represents an additional, non-quantified advantage of plant oil substitution that is not captured by the static cost comparison in Table 6. Because these estimates are based on indicative rather than supplier-verified prices, they should be interpreted as illustrating the likely direction and approximate scale of the cost advantage rather than as a precise costing for any specific production context; the feed conversion ratio itself, which does not depend on price assumptions, was not compromised by either oil. To assess the robustness of these cost estimates to fish oil price fluctuations, we recalculated RCS for the CO and SO diets under fish oil prices ranging from −20% to +20% of the base price (2.20 USD · kg⁻¹), holding all other ingredient prices constant. Because the CO and SO diets contain no added fish oil, their own feed cost is unaffected by this parameter; only the FO control’s feed cost, and therefore the denominator of the RCS calculation, changes. Across this range, RCS for the CO diet varied from approximately 8.8% (at −20% fish oil price) to 17.7% (at +20%), and for the SO diet from approximately 7.5% to 16.6%, confirming that the direction and approximate magnitude of the cost advantage is robust to plausible fish oil price fluctuations (Suppl. material 1: table SS3). Combined with canola oil’s substantially better retention of the muscle n-3/n-6 ratio relative to safflower oil, this cost pattern points to canola oil specifically, rather than plant oil substitution in general, as the more defensible single recommendation arising from this study; safflower oil’s cost advantage appears comparable, but its effect on the muscle n-3/n-6 ratio is sufficiently large that its use as a sole substitute, rather than as one component of a blend or a short-duration treatment, would require an explicit trade-off decision regarding product nutritional quality that does not arise with canola oil.

Limitations of the study. Several limitations of the present study should be acknowledged. First, while the growth performance and fatty acid deposition data provide a robust basis for evaluating canola and safflower oils as fish oil substitutes, the study did not include molecular analyses (hepatic gene expression, enzyme activities, or fatty acid desaturase/elongase assays) that would be required to elucidate the mechanistic basis of the organ-index and non-additive blend effects observed. Such analyses represent a logical next step for this species. Additionally, this study did not assess intestinal histology, inflammatory markers, or other indices of metabolic stress that have been reported following complete or high-level plant oil substitution in some fish species, including shortened intestinal folds in Atlantic salmon (Moldal et al. 2014) and altered pro-inflammatory cytokine expression in gilthead sea bream (Montero et al. 2010). Given that such effects could occur independently of the growth, feed conversion, and organ-index outcomes reported here, this represents an important gap in the present characterization of canola and safflower oil as fish oil substitutes in Black Sea trout. Second, the study did not assess oxidative stability, sensory quality, or shelf-life of fillets from fish fed the experimental diets, which are commercially relevant parameters that may differ between fish oil- and plant oil-fed fish due to differences in fatty acid unsaturation and endogenous antioxidant content. Third, although three replicate tanks per treatment (n = 3) are standard for this type of feeding trial and were sufficient to detect statistically significant differences in several parameters, the limited replication reduces statistical power for detecting more subtle treatment effects. Fourth, as noted above, the feeding trial was conducted in 2015, and the economic analysis uses approximate 2024 ingredient prices rather than prices contemporaneous with the trial. Finally, the study was conducted on juvenile fish under controlled tank conditions over 90 days; extrapolation to commercial-scale production, larger fish, or different environmental conditions should be made with caution. These limitations do not undermine the central conclusions of the study but define the scope of inference and identify priorities for future research.

This study indicates that canola oil is the more favorable single substitute for fish oil in Black Sea trout grow-out diets under the conditions tested, offering growth equivalence, favorable hepatic profile, superior n-3/n-6 ratio retention (0.32 vs. 0.04 in safflower oil-fed fish), and an estimated 13.46% feed cost saving. Safflower oil achieves a comparable estimated cost saving (12.29%) but causes a disproportionately large collapse in the muscle n-3/n-6 ratio, restricting its practical use to blended formulations (≤50% of added oil) or short-duration feeding strategies rather than as a stand-alone replacement.

More broadly, we demonstrate that oil blends do not behave as linear intermediates between their single-oil components—a finding that challenges a common assumption in commercial feed formulation. The significantly elevated hepatosomatic index observed in the safflower-canola blend relative to either oil alone, and the non-additive muscle fatty acid class values reported for several binary blends, both underscore the need for empirical testing of each oil combination in each target species.

For Black Sea trout producers, we recommend canola oil as the primary lipid source in grow-out diets, as it offers the optimal balance of cost saving and product quality among the oils tested. Where safflower oil is used, it should be restricted to no more than 50% of the added oil fraction in blended formulations, given the disproportionate depletion of the muscle n-3/n-6 ratio observed when it is used as a sole substitute. For premium markets requiring elevated muscle EPA and DHA, a pre-harvest fish oil finishing period of 4–6 weeks is recommended to restore n-3 LC-PUFA levels.

Future research should prioritize characterizing the LC-PUFA biosynthetic capacity of S. labrax using stable isotope tracers, testing the hepatic and enzymatic basis of the blend-specific HSI response through targeted gene expression studies, evaluating intestinal histology, inflammatory gene expression, and other indices of metabolic stress in Black Sea trout fed canola and safflower oil-based diets, particularly at complete substitution levels, alongside growth and fatty acid outcomes, and extending the feed-cost analysis to a full cost-benefit framework accounting for the market value of the final product.