Introduction
Aquatic products are highly desirable in the human diet, as they provide essential omega-3 fatty acids, high-quality proteins, vitamins, and minerals. However, the growing commercialization of products derived from fisheries and aquaculture creates increasing opportunities for misrepresentation and fraud. Seafood is among the most vulnerable food categories globally, due to complex supply chains, high economic value, and frequent substitution and mislabeling. Aquaculture continues to grow as a sustainable source of animal protein, but robust tools are needed to verify the origin and production method of farmed and wild products. This study addresses these challenges by integrating stable isotope ratio analysis (SIRA), multi-elemental fingerprinting, and fatty acid profiling to differentiate between wild and farmed Gilthead seabream (Sparus aurata), determine geographical origin, and assess product quality based on fatty acid composition and the carbon stable isotope ratios of individual fatty acids. Obtained data are added into the IsoFoodTrack reference database which serves as a centralized repository for storing analytical data, supporting traceability and authenticity verification and food fraud prevention. To evaluate sample classification and discrimination by origin and production method, multivariate statistical models such as Orthogonal Partial Least Squares Discriminant Analysis (OPLS-DA) and Data-Driven Soft Independent Modelling of Class Analogy (DD-SIMCA) were applied, demonstrating the effectiveness of chemometric approaches in seafood authentication.
Materials and Methods
A total of 163 authentic and commercial samples of seabream were collected from various marine environments, including the Mediterranean Sea, Adriatic Sea, and the Atlantic Ocean. The stable isotope ratios of light elements (δ¹³C, δ¹⁵N, δ³⁴S) were measured in defatted muscle tissues using IsoPrime100 – Vario PYRO Cube Isotope Ratio Mass Spectrometer (IRMS), while the measurements of oxygen (δ18O) isotopic composition in isolated water from the tissue were determined by IsoPrime MultiFlow system coupled to IRMS. Multi-elemental profiling of 30 elements was conducted by Inductively Coupled Plasma Mass Spectrometry (ICP-MS). Fatty acid methyl ester composition was determined by Gas Chromatography-Mass Spectrometry Detection (GC-MSD), while carbon isotope ratios of individual fatty acids were analyzed by Gas Chromatography–Combustion–Isotope Ratio Mass Spectrometry (GC-C-IRMS).
Results and discussion
Stable isotope ratios of δ¹³C, δ¹⁵N, and δ³⁴S effectively differentiated wild from farmed Sparus aurata, with wild samples showing enriched δ¹³C and δ¹⁵N values, consistent with a marine-based diet and higher trophic level. Elemental signatures, complemented the isotopic data and further supported the differentiation of geographic origin and production type. The most powerful variables for distinguishing samples from five different geographical locations were δ¹⁸O, K, P, Rb, δ¹³C, δ³⁴S, Mg, Zn, δ¹⁵N, S, Co, Hg, and Se. Classification models using OPLS-DA achieved 94% accuracy for geographic origin and 100% accuracy for production method differentiation. The DD-SIMCA model was used for class modeling and verification of Portuguese-origin samples, achieving 96% accuracy, with 93% sensitivity and 98% specificity.
Fatty acid profiling revealed significantly higher concentrations of long-chain ω-3 polyunsaturated fatty acids (EPA and DHA) in wild seabream, as expected based on their natural diet and previous studies. EPA and DHA levels in wild fish reached 7.4% and 17.1%, respectively, compared to 2.1% and 5.3% in farmed seabream. These differences reflect dietary and environmental influences, including feed composition and production method. In addition, preliminary results from carbon isotope ratios of individual fatty acids suggest further potential for differentiating production origin and identifying biogenetic sources, highlighting the value of combining GC-MSD and GC-C-IRMS for assessing nutritional quality and feeding patterns.
Conclusion
This study demonstrates the effectiveness of an integrated analytical approach for seafood authentication and quality assessment. The resulting data contribute to the IsoFoodTrack database, supporting the development of reliable traceability systems and reinforcing efforts to prevent seafood fraud across the supply chain.
Acknowledgements
This work was supported by the Slovenian Research Agency within Programmes P1-0143, Young Researcher grant programme (57078), Horizon Europe FishEUTrust project (No. 101060712) and METROFOOD-SI (No. 871083).