P. Smíd, I. Doskocil, B. Lampova, A. Kopec
Changes of omega-3 and omega-6 fatty acids content in sardines and sprats after heat treatment

Fatty acids play a major role in human nutrition and diet. The correct fatty acid composition is important and leads to beneficial health effects. Among fatty acids, omega-3 and omega-6 are most often dealt with. Their ratio in diet should be 4:1 (n-6:n-3) according to WHO. Those fatty acids are essential and play a key role in inflammatory processes, promoting cognitive functions and cardiovascular system health. Sardines and sprats are rich in fat and so in omega-3 and omega-6 fatty acids. Heat treatment can affect lipid peroxidation, leading to loss of fatty acids and significantly changing the omega-3 and 6 ratio. We tested the four most common heat treatment methods baking, deep frying, steaming, and boiling. Major omega-3 and 6 fatty acids were observed - linoleic acid, gamma-linoleic acid, arachidonic acid, linolenic acid, eicosatetraenoic acid and docosahexaenoic acid. Samples were from the Baltic and Mediterranean Sea. Before analysis, whole fish bodies were lyophilized and homogenized. From 1g of dry samples, fat was extracted using syringes and a vacuum pump with 16 ml of ethanol:acetone:hexane solution (1:1:2; V:V:V). Then solution was evaporated using rotary vacuum evaporator. Then 40 µl of fat was taken, and 0,5 ml of methanol HPLC and 0,5 ml natrium methanolate solution was added. Prepared tube was shaken and put in a water bath with 75-80 °C water and tempered. After 3 mins tube was shaken and cooled with cold water. Then 1,5 ml of hexane and saturated sodium chloride solution was added and carefully shaken. Samples were centrifuged for 5 mins at 5000 rpm to separate the organic phase. Finally, 400 µl of the sample was taken for GC/FID analysis. Results vary greatly for individual fatty acids, but in general, cooked sardines had almost 10 times lower content of n-6 fatty acids and 3 times lower content of n-3 fatty acids. The most appropriate heat treatment for sardines is baking, where losses are lowest. As for sprats, heat treatment increases the content of n-3 and n-6 fatty acids, whereby steamed samples had the highest content. Fried samples are distorted by the frying medium. This research was funded by the Grant Agency of the Czech Republic, grant no. GA 21-42021L. And the National Science Centre Poland, grant no. 2020/39/I/NZ9/02959.

T. Diallo, Y. Makni, A. Lerebours, H. Thomas, T. Guérin, J. Parinet
Bivalves as sentinel species to Follow-up the eco-exposome: The sample preparation as a major challenge

In recent decades, coastal waters have been subjected to a variety of contaminants from urban, industrial and agricultural activities. Numerous persistent chemicals that reach the estuarine environment via rivers, wastewater treatment plants and watershed leaching accumulate in seawater and/or aquatic organisms. The Pertuis-Charentais area, in southwest France, is one of them. This area is a transition zone between the Atlantic Ocean and the estuaries of the rivers Sèvre, Charente and Seudre. This region produces 22% of French shellfish production and France is the second largest producer of bivalves in Europe (Diallo et al., 2022). The mortality observed since 2008 in mussels and oysters, sentinel species of environmental quality, suggests a deterioration in water quality, but the precise causes are not yet identified. In this context, the AMPHIBIE project, funded by the ANR JCJC "AlimOnic" , aims to characterise the contamination by pesticides, veterinary drugs, pharmaceuticals and plastic additives on a large scale in bivalves through active biomonitoring for one year. For this purpose, a comprehensive screening method based on LC-HRMS was developed and validated. Different extraction and purification procedures (QuEChERS/QuPPe) were optimised with pooled seafood samples (bivalves, fishes and crustaceans) using a representative mixture of 300 contaminants (pesticides and veterinary drugs) from different families, with very distinct and large physicochemical properties. To simplify the optimisation of the extraction and purification procedures, the design of experiments (DoE) was used (Taguchi orthogonal array). The screening method was validated according to the EU SANTE/11312/2021 guideline, by extending the number of contaminants up to 850 (pesticides and veterinary drugs). For each contaminant, the screening detection limits (SDLs) and the limits of identification (LOI) were established in seafood samples. The method was also tested for the analysis of other organic contaminants such as pharmaceutical to demonstrate its versatility by suspect screening in non-spike seafood samples. Finally, the validated methods were applied to biomonitoring real samples of mussels and oysters collected at different times in different sites (with different mortality rates) in southwestern France to try to find markers of exposure and effect of high mortality of bivalves.

A. Riccaboni, S. Cresti, C. Tozzi, V. Miceli, C. Zoani
Agrifood Living Labs: the METROFOOD-IT Living Labs

Agrifood is one of the major sectors contributing to pressures on planetary boundaries by affecting biogeochemical cycles, climate, ecosystems, and biodiversity with about a third of greenhouse gas (GHG) emissions at the global level (FAO,2018). A radical transformation of the traditional way of production, processing, distribution, and consumption of food is needed. Place-based Living Labs can facilitate the synergies of the major players and the connections of projects, initiatives, and best practices, as well as enhance collaboration between sectors, such as local food, transport, and energy (Bulkeley et al., 2016). Agrifood Living Labs are a new type of research and collaboration centered organisations based on co-creation, aiming at promoting innovation in the agrifood sector (Mulvenna et al., 2011). METROFOOD-IT - the Italian Research Infrastructure for Metrology and Open Access Data in support to the Agrifood, in relation to the ESFRI METROFOOD-RI for the domain Health and Food - will run Living Labs as a co-creative space for codesigning, experiencing, and assessing new solutions in support to the agrifood systems, where users will be also directly involved in evaluating new ideas, concepts, and technological solutions. The Living Lab on circular bioeconomy and industrial symbiosis, hosted at the ENEA Research Center of Brindisi (Italy), includes access to a demonstrator that will showcase how biomolecules can be recovered from wastes generated by the agrifood chain to generate new production processes. The innovations showcased will be inspired by the "zero waste at the end of the process" objective, placing sustainability at the centre of the strategies and thus promoting "greening" processes and systemic interventions aimed at spreading eco- innovative models on the market. Benefits will be related also to the reduction of the environmental impact of plastics and pollutants from the dairy industry integrating it with territorial and socio-economic indicators. The Agrifood FabLab hosted by the Santa Chiara Lab of the University of Siena (Italy) focuses on sustainability of the agrifood systems by mobilising all the agrifood system actors, including food businesses, local authorities and policy makers, investors, entrepreneurs, consumers, and stakeholders, as wellas transferring the technology in their production processes. The Agrifood FabLab represents an innovative environment focused on user communities embedded within "real life" situations and environments.

S. Ciano, E. Van Hoeck, M. Andjelkovic, N. Waegeneers, S. Goscinny
Dietary exposure of Sorbates (E 200 202) and Benzoates (E 210 213) for the Belgian population

Sorbates (E 200 202) and benzoates (E 210 - 213) are commonly used food additives (FAs). They act as bacteriostatic and fungistatic, and they ensure product quality. Also, they contribute to reducing food waste by extending the shelf-life of perishable items. However, Regulation (EC) No 1333/2008 requires the Member States to monitor the consumption and use of FAs using a risk-based approach and communicate the results to the European Commission and the National authorities. So, this study assessed the analytical concentration of sorbates (SA) and benzoates (BA) in food and beverages from the Belgian market. Subsequently, the exposure of different consumer populations to these FAs was estimated. Three matrix-matched analytical methods were developed using ion chromatography coupled with conductivity detection. The methods were validated in-house and applied to 387 samples covering 32 Food (sub)Categories. SA and BA were mentioned on the label of 367 and 111 samples, respectively. SA were quantified in 97% of these samples, while BA were present in 86%. The concentrations varied widely due to the nature of the food/beverage and the specific Maximum Permitted Levels (MPLs) defined by Regulation (EC) No 1333/2008. MPLs were exceeded in 17 samples, while composite foods showed average concentrations higher than in previous studies. A refined exposure assessment revealed no risk related to dietary exposure to SA or BA in 3 Belgian population groups (children, adolescents and adults). The occurrence data from the chemical analyses were combined with consumption data from ther most recent Belgian food consumption survey reflecting the consumer's consumption patterns and frequencies. Mean exposure estimates ranged from 8 to 19% of the Allowed Daily Intakes (ADIs) (i.e. 11 and 5 mg/kg bw per day for SA and BA, respectively), and 95th percentile exposures ranged from 24 to 36% of the ADIs. Flavoured drinks were the major contributing food group, accounting on average for 21 to 39% of the exposure to SA and 31 to 44% of the exposure to BA (depending on the population group). In conclusion, although a few products surpassed the allowed levels of targeted FAs, the estimated risk related to dietary exposure to SA or BA for Belgian children, adolescents or adults was low

M. Vankoningsloo, M. Andjelkovic, D. Stanic-Vucinic, V. Jovanovic, J. Mutic, T. Mutic, J. Acimovic, B. Andjelkovic, A. Rajkovic, T. Cirkovic Velickovic
Dietary exposure to microplastic via shellfish and the importance of the edible shellfish tissue measurements

Shellfish are believed to be the major food source of micro- and nano plastics (MNPs) originating from the food chain. Therefore, consumption of shellfish carries a risk of exposure to MNPs and their cargo (organic and inorganic pollutants and pathogens), but also allergens as MNP cargo, potentially influencing sensitization and allergic response. To estimate the exposure to MNPs it is necessary to have developed and standardised analytical methods. This will consequently lead to comparable exposure assessment and risk characterisation for humans. Particularly in this study we are pointing out the impact of the metrics selection (shellfish tissue quantity) on the estimation of exposure assessment. Shellfish (clams (n= 83) and mussels (n=47)) were collected from food markets in Belgium, Croatia, Serbia and South Korea. MPs were isolated from shellfish samples using an optimized digestion protocol followed by counting and characterization by µFTIR. Number of MPs was expressed per individual and per gram of soft (edible) wet tissue (EWT). Hence, before digestion, mass of total shell content (including EWT and intra-valvular liquid) was measured for every individual. EWT represented around 50-70% of the whole shellfish content. A range of 0.13-0.20 MPs per g of shell content vs 0.19-0.33 MPs per g of EWT was determined. Based on the average adult consumption of 225 g of mussels [1] and combined with the quantified MPs in this study, the exposure estimates (deterministic approach) would range from 0.41- 0.64 MP per kg bw/day expressed per total shell content to 0.61-1.06 MP per kg bw/day when expressed per EWT. In other words, standardization of the exposure metrics may reduce uncertainty by 30-40% resulting in a more relevant and less biased outcome. Moreover, preliminary hazard identification had shown that polystyrene and polypropylene were the most frequent types of MPs, and prevalence data showed presence of MP in about 50% of tested samples. Provided all of the above, it is critical that the number of MP particles is isolated correctly and efficiently. The notable observation is that MP particles could be expressed per g of wet tissue (consumed part) which represents 50-70% of the shellfish content and it may impact the exposure metrics by 30-40%.